[3.2.0] Heterocyclic compounds and methods of using the same

ABSTRACT

Compounds of Formulae I-VI and derivatives thereof having anti-cancer, anti-inflammatory, and anti-microbial properties and to compositions that include one or more of compounds and their derivatives or analogs having anti-cancer, anti-inflammatory and anti-microbial properties are disclosed. Pharmaceutical compositions comprising such compounds and methods of treating cancer, inflammatory conditions, and microbial infections with the disclosed compounds or the disclosed pharmaceutical compositions are also disclosed.

This application is a continuation of U.S. application Ser. No.11/118,260, entitled [3.2.0] HETEROCYCLIC COMPOUNDS AND METHODS OF USINGTHE SAME, filed on Apr. 29, 2005 which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 60/567,336, filed on Apr.30, 2004; 60/580,838, filed on Jun. 18, 2004; 60/591,190, filed on Jul.26, 2004; 60/627,462, filed on Nov. 12, 2004; 60/644,132, filed on Jan.13, 2005; and 60/659,385, filed on Mar. 4, 2005; each of which isentitled [3.2.0] HETEROCYCLIC COMPOUNDS AND METHODS OF USING THE SAME;and each of which is incorporated herein by reference in its entiretyincluding any and all drawings.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to certain compounds and to methods forthe preparation and the use of certain compounds in the fields ofchemistry and medicine. More specifically, the present invention relatesto compounds and procedures for making and using compounds that areuseful as anti-cancer, anti-inflammatory, and anti-microbial agents, andrelates to pharmaceutical dosage forms comprising such compounds.

2. Description of the Related Art

Cancer is a leading cause of death in the United States. Despitesignificant efforts to find new approaches for treating cancer, theprimary treatment options remain surgery, chemotherapy and radiationtherapy, either alone or in combination. Surgery and radiation therapy,however, are generally useful only for fairly defined types of cancer,and are of limited use for treating patients with disseminated disease.Chemotherapy is the method that is generally useful in treating patientswith metastatic cancer or diffuse cancers such as leukemias. Althoughchemotherapy can provide a therapeutic benefit, it often fails to resultin cure of the disease due to the patient's cancer cells becomingresistant to the chemotherapeutic agent. Due, in part, to the likelihoodof cancer cells becoming resistant to a chemotherapeutic agent, suchagents are commonly used in combination to treat patients.

Similarly, infectious diseases caused, for example, by bacteria, fungiand protozoa are becoming increasingly difficult to treat and cure. Forexample, more and more bacteria, fungi and protozoa are developingresistance to current antibiotics and chemotherapeutic agents. Examplesof such microbes include Bacillus, Leishmania, Plasmodium andTrypanosoma.

Furthermore, a growing number of diseases and medical conditions areclassified as inflammatory diseases. Such diseases include conditionssuch as asthma to cardiovascular diseases. These diseases continue toaffect larger and larger numbers of people worldwide despite newtherapies and medical advances.

Therefore, a need exists for additional chemotherapeutics,anti-microbial agents, and anti-inflammatory agents to treat cancer,inflammatory diseases and infectious disease. A continuing effort isbeing made by individual investigators, academia and companies toidentify new, potentially useful chemotherapeutic and anti-microbialagents.

Marine-derived natural products are a rich source of potential newanti-cancer agents and anti-microbial agents. The oceans are massivelycomplex and house a diverse assemblage of microbes that occur inenvironments of extreme variations in pressure, salinity, andtemperature. Marine microorganisms have therefore developed uniquemetabolic and physiological capabilities that not only ensure survivalin extreme and varied habitats, but also offer the potential to producemetabolites that would not be observed from terrestrial microorganisms(Okami, Y. 1993 J Mar Biotechnol 1:59). Representative structuralclasses of such metabolites include terpenes, peptides, polyketides, andcompounds with mixed biosynthetic origins. Many of these molecules havedemonstrable anti-tumor, anti-bacterial, anti-fungal, anti-inflammatoryor immunosuppressive activities (Bull, A. T. et al. 2000 Microbiol MolBiol Rev 64:573; Cragg, G. M. & D. J. Newman 2002 Trends Pharmacol Sci23:404; Kerr, R. G. & S. S. Kerr 1999 Exp Opin Ther Patents 9:1207;Moore, B. S 1999 Nat Prod Rep 16:653; Faulkner, D. J. 2001 Nat Prod Rep18:1; Mayer, A. M. & V. K. Lehmann 2001 Anticancer Res 21:2489),validating the utility of this source for isolating invaluabletherapeutic agents. Further, the isolation of novel anti-cancer andanti-microbial agents that represent alternative mechanistic classes tothose currently on the market will help to address resistance concerns,including any mechanism-based resistance that may have been engineeredinto pathogens for bioterrorism purposes.

SUMMARY OF THE INVENTION

Disclosed herein are compounds, pharmaceutical compositions comprisingsuch compounds, uses of such compounds and compositions, and methods forthe preparation of such compounds, having the structure of FormulaeI-VI:

Some embodiments relate to compounds having the structure of Formula I,and pharmaceutically acceptable salts and pro-drug esters thereof:

wherein the dashed line indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ can be separately selectedfrom the group consisting of a hydrogen, a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl(including, for example, cyclohexylcarbinol, cycloalkenyl, alkoxy,cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl,amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl,cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy,cyano, thio, sulfoxide, sulfone, sulfonate esters, thiocyano, boronicacids and esters, and halogenated alkyl including polyhalogenated alkyl.n is equal to 1 or 2, and if n is equal to 2, then R₁ can be the same ordifferent;

wherein R₂, can be selected from the group consisting of hydrogen, ahalogen, mono-substituted, poly-substituted or unsubstituted variants ofthe following residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl (including, for example,cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl;

wherein R₃ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl; whereineach of E₁, E₂, E₃ and E₄ can be a substituted or unsubstitutedheteroatom; and with the proviso that Formula I is not Compound II-16 orCompound II-17.

In some embodiments, R₂ is not cyclohex-2-enyl carbinol when one of theR₁ substituents is ethyl or chloroethyl and R₃ is methyl.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

R₂ can be a formyl. For example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

As another example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

As a further example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

Also, R₂ can be a cyclohexenylmethylene or 3-methylenecyclohexene.

For example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

R₂ can be a cyclohexylalkylamine, a C-Cyclohexyl-methyleneamine, or acyclohexanecarbaldehyde O-oxime.

For example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I;and wherein R₉ is selected from the group consisting of hydrogen,substituted or unsubstituted variants of the following: alkyl, acyl,aryl and heteroaryl.

Furthermore, R₂ can be a cycloalkylacyl.

As an example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, I, and Br.

Further embodiments relate to compounds having the structure of FormulaII, and pharmaceutically acceptable salts and pro-drug esters thereof:

wherein the dashed lines indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ can be separately selectedfrom the group consisting of a hydrogen, a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy,nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thiocyano,boronic acids and esters, and halogenated alkyl includingpolyhalogenated alkyl where n is equal to 1 or 2, and if n is equal to2, then R₁ can be the same or different;

wherein R₃ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl;

wherein R₄ can be separately selected from the group consisting of ahydrogen, a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl, and m is equal to 1or 2, and if m is equal to 2, then R₄ can be the same or different;

wherein each of E₁, E₂, E₃, E₄ and E₅ can be a substituted orunsubstituted heteroatom; and

with the proviso that Formula II is not Compound II-16 or CompoundII-17. Some embodiments include the proviso that one R₄ is notcyclohex-2-enyl, if the other R₄ is hydrogen and if m and n are equal to2, and if R₃ is methyl, and if one of the substituents R₁ is ethyl orchloroethyl while the other R₁ is hydrogen.

As an example, E₅ can be selected from the group consisting of OH, O, S,N, NH, NH₂, NOH, NHOH, OR₁₀, SR₁₁, NR_(12,) and NHOR₁₃, wherein each ofR₁₀, R₁₁, R₁₂, and R₁₃ can be separately selected from the groupconsisting of hydrogen, and a substituted or unsubstituted alkyl, acyl,aryl, and heteroaryl.

n can be equal to 1 or 2, and where n is equal to 2, at least one R₁ canbe CH₂CH₂X, wherein X can be selected from the group consisting of H, F,Cl, Br, and I.

As another example, R₃ can be methyl. Furthermore, E₅ can be OH. Each ofE₁, E₃ and E₄ can be O and E₂ can be NH. At least one R₄ can be acycloalkane or cycloalkene, for example. In another example, n is equalto 2 and at least one of the R₁ substituents is hydrogen and the otherR₁ substituent is CH₂CH₂X, wherein X is selected from the groupconsisting of H, F, Cl Br, and I; wherein at least one R₄ is cyclohexaneor cyclohexene; wherein E₅ is OH; wherein R₃ is methyl; and wherein eachof E₁, E₃ and E₄ is O and E₂ is NH.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

As an example, the structure may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br and I.

At least one R₄ can be a di-substituted cyclohexane, for example,

wherein R₈ is selected from the group consisting of H, F, Cl, Br and I.

R₆ and R₇ both can be OH.

At least one R₄ can be a 7-oxa-bicyclo[4.1.0]hept-2-yl, for example:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

Also, at least one R₄ can be a substituted or an unsubstituted branchedalkyl, for example the following compound:

wherein R₈ is selected from the group consisting of H, F, Cl, Br and I.

Furthermore, at least one R₄ can be a cycloalkyl and E₅ can be anoxygen.

As an example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, I, and Br.

As a further example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I;and wherein R₉ can be selected from the group consisting of H,substituted and unsubstituted alkyl, substituted and unsubstituted aryl,and substituted and unsubstituted heteroaryl.

In still a further example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

Also, at least one R₄ can be a cycloalkyl and E₅ can be NH₂. Forexample, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

In still a further example, the compound can be a prodrug ester orthioester, for example, the compound may be:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.

Other embodiments relate to compounds having the structure of FormulaIII, and pharmaceutically acceptable salts and pro-drug esters thereof:

wherein the dashed lines indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ can be separately selectedfrom the group consisting of a hydrogen, a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy,nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thiocyano,boronic acids and esters, and halogenated alkyl includingpolyhalogenated alkyl, and n is equal to 1 or 2, and if n is equal to 2,then R₁ can be the same or different;

wherein R₄ can be separately selected from the group consisting ofhydrogen, a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl, and m is equal to 1or 2, and if m is equal to 2, then R₄ can be the same or different; andwherein each of E₁, E₂, E₃, E₄ and E₅ is a substituted or unsubstitutedheteroatom; and with the proviso that Formula III is not Compound II-16or Compound II-17. Some embodiments include the proviso that one R₄ isnot cyclohex-2-enyl, if the other R₄ is hydrogen and if m and n areequal to 2 and if one of the substituents R₁ is ethyl or chloroethylwhile the other R₁ is hydrogen.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

Still further embodiments relate to compounds having the structure ofFormula IV, and pharmaceutically acceptable salts and pro-drug estersthereof:

wherein the dashed lines indicate that the designated bond is either asingle bond or a double bond, and wherein R₁ can be separately selectedfrom the group consisting of a hydrogen, a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy,nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thiocyano,boronic acids and esters, and halogenated alkyl includingpolyhalogenated alkyl, and n is equal to 1 or 2, and if n is equal to 2,then R₁ can be the same or different;

wherein R₃ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl;

wherein R₅ can be separately selected from the group consisting of ahydrogen, a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, oxy, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl, wherein m is 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or 11 and if m is more than 1, then R₅ can bethe same or different; and where the substituents R₅ can form a ring;and wherein each of E₁, E₂, E₃, E₄ and E₅ is a substituted orunsubstituted heteroatom.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

Other embodiments relate to compounds having the structure of Formula V,and pharmaceutically acceptable salts and pro-drug esters thereof:

wherein the dashed line indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ can be selected from thegroup consisting of a hydrogen, a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy,nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thiocyano,boronic acids and esters, and halogenated alkyl includingpolyhalogenated alkyl, and n is equal to 1 or 2, and if n is equal to 2,then R₁ can be the same or different;

wherein R₅ is separately selected from the group consisting of ahydrogen, a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, oxy, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl, wherein m is 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or 11, and if m is more than 1, then R₅ can bethe same or different; and where the substituents R₅ can form a ring;and wherein each of E₁, E₂, E₃, E₄ and E₅ is a substituted orunsubstituted heteroatom.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

Other embodiments relate to compounds having the structure of FormulaVI, and pharmaceutically acceptable salts and pro-drug esters thereof:

wherein R₁ can be separately selected from the group consisting of amono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, phenyl, cycloalkylacyl, alkylthio,arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfoneboronic acidesters, and halogenated alkyl including polyhalogenated alkyl. n isequal to 1 or 2, and if n is equal to 2, then R₁ can be the same ordifferent;

wherein R₂, can be selected from the group consisting of hydrogen, ahalogen, mono-substituted, poly-substituted or unsubstituted variants ofthe following residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl (including, for example,cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl;

wherein R₃ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl; whereineach of E₁, E₂, E₃ and E₄ can be a substituted or unsubstitutedheteroatom.

In some embodiments, R₂ is not cyclohex-2-enyl carbinol when one of theR₁ substituents is ethyl or chloroethyl and R₃ is methyl.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred.

wherein R₁₄ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,thioesters, sulfoxide, sulfone, sulfonate esters, thiocyano, andhalogenated alkyl including polyhalogenated alkyl.

In some embodiments, preferably R₁₄ is an alkylthiol or substitutedalkylthiol, and E₃ is an oxygen.

Some exemplary embodiments relate to compounds of Formula VI that havethe following structure, and which are referred to as Formula VI-I:

wherein R₁ can be separately selected from the group consisting of amono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, phenyl, cycloalkylacyl, alkylthio,arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, sulfonateesters, thiocyano, boronic acid esters, and halogenated alkyl includingpolyhalogenated alkyl. n is equal to 1 or 2, and if n is equal to 2,then R₁ can be the same or different;

wherein R₃ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl; whereineach of E₁, E₂, E₃ and E₄ can be a substituted or unsubstitutedheteroatom.

wherein R₅ can be separately selected from the group consisting of ahydrogen, a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, oxy, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl, wherein m is 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or 11 and if m is more than 1, then R₅ can bethe same or different; and where the substituents R₅ can form a ring;and wherein each of E₁, E₂, E₃, E₄ and E₅ is a substituted orunsubstituted heteroatom.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred.

wherein R₁₄ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,thioesters, sulfoxide, sulfone, sulfonate esters, thiocyano, andhalogenated alkyl including polyhalogenated alkyl.

In some embodiments, preferably R₁₄ is an alkylthiol or substitutedalkylthiol, and E₃ is an oxygen.

For example, the compound has the following structure VI-1A:

Exemplary stereochemistry can be as follows:

For example, an exemplary compound of Formula VI has the followingstructure and stereochemistry VI-1B:

Another example, the compound of Formula VI has the following structureand stereochemistry VI-1C:

Some embodiments relate to pharmaceutical compositions that include acompound as described herein. The pharmaceutical compositions mayfurther include an anti-microbial agent.

Other embodiments relate to methods of treating cancer. The methods mayinclude, for example, administering a compound or composition asdescribed herein, and pharmaceutically acceptable salts and pro-drugesters thereof The methods may further include the steps of: identifyinga subject that would benefit from administration of an anticancer agent;and performing the method on the subject. The cancer may be, forexample, a multiple myeloma, a colorectal carcinoma, a prostatecarcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, anovarian carcinoma, a melanoma, and the like. The cancer can be adrug-resistant cancer, and the drug-resistant cancer may display atleast one of the following: elevated levels of the P-glycoprotein effluxpump, increased expression of the multidrug-resistance associatedprotein 1 encoded by MRP1, reduced drug uptake, alteration of the drug'starget or increasing repair of drug-induced DNA damage, alteration ofthe apoptotic pathway or the activation of cytochrome P450 enzymes. Asan example, the drug-resistant cancer can be a sarcoma.

Still further embodiments relate to methods of inhibiting the growth ofa cancer cell. The methods can include, for example, contacting a cancercell with a compound or composition as described herein, andpharmaceutically acceptable salts and pro-drug esters thereof The cancercell may be, for example, a multiple myeloma, a colorectal carcinoma, aprostate carcinoma, a breast adenocarcinoma, a non-small cell lungcarcinoma, an ovarian carcinoma, a melanoma, and the like.

Other embodiments relate to methods of inhibiting proteasome activitycomprising the step contacting a cell with a compound or composition asdescribed herein, and pharmaceutically acceptable salts and pro-drugesters thereof

Further embodiments relate to methods of inhibiting NF-κB activation.The methods can include, for example, the step contacting a cell with acompound or composition as described herein, and pharmaceuticallyacceptable salts and pro-drug esters thereof

Still other embodiments relate to methods for treating an inflammatorycondition. The methods may include, for example, administering aneffective amount of a compound or composition as described herein to apatient in need thereof The inflammatory condition may be, for example,rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, stroke,myocardial infarction, and the like.

Some embodiments relate to methods for treating a microbial illnesswhich can include administering an effective amount of a compound orcomposition as described herein to a patient in need thereof Themicrobial illness maybe caused, for example by B. anthracis, Plasmodium,Leishmania, and Trypanosoma.

Other embodiments relate to uses of one or more compounds of Formulae I,II, III, IV, V, or VI, and pharmaceutically acceptable salts andpro-drug esters thereof in the treatment of a cancer, an inflammatorycondition, or a microbial infection. The one or more compounds is one ormore of a compound of any of the compounds as described herein, andpharmaceutically acceptable salts and pro-drug esters thereof The cancercan be, for example a multiple myeloma, a colorectal carcinoma, aprostate carcinoma, a breast adenocarcinoma, a non-small cell lungcarcinoma, an ovarian carcinoma or a melanoma. Also, the cancer can be adrug-resistant cancer, for example, one that displays at least one ofthe following: elevated levels of the P-glycoprotein efflux pump,increased expression of the multidrug-resistance associated protein 1encoded by MRP1, reduced drug uptake, alteration of the drug's target orincreasing repair of drug-induced DNA damage, alteration of theapoptotic pathway and the activation of cytochrome P450 enzymes. Theinflammatory condition can be, for example, rheumatoid arthritis,asthma, multiple sclerosis, psoriasis, stroke, or myocardial infarction.The microbial infection can be, for example, caused by B. anthracis,Plasmodium, Leishmania, or Trypanosoma.

Still further embodiments relate to uses of one or more compounds ofFormulae I, II, III, IV, V, or VI as described herein, andpharmaceutically acceptable salts and pro-drug esters thereof, for theinhibition of angiogenesis, inhibition of a proteasome activity, orinhibition of NF-κB activation.

Some embodiments relate to uses of a compound of Formula I, II, III, IV,V, or VI, and pharmaceutically acceptable salts and pro-drug estersthereof, in the preparation of medicament for the treatment of a cancer,an inflammatory condition, or a microbial infection or for theinhibition of angiogenesis, a proteasome activity, or NF-κB activation.The uses can further include the use of a chemotherapeutic agent, ananti-angiogenic agent, an anti-inflammatory agent, or a proteasomeinhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form part ofthe specification, merely illustrate certain preferred embodiments ofthe present invention. Together with the remainder of the specification,they are meant to serve to explain preferred modes of making certaincompounds of the invention to those of skilled in the art. In thedrawings:

FIG. 1 depicts the ¹H NMR spectrum of a compound having structureFormula II-2.

FIG. 2 depicts the mass spectrum of a compound having structure FormulaII-2.

FIG. 3 depicts the ¹H NMR spectrum of a compound having structureFormula II-3.

FIG. 4 depicts the mass spectrum of a compound having structure FormulaII-3.

FIG. 5 depicts the ¹H NMR spectrum of a compound having structureFormula II-4.

FIG. 6 depicts the mass spectrum of a compound having structure FormulaII-4.

FIG. 7 depicts the ¹H NMR spectrum of a compound having structureFormula II-5A.

FIG. 8 depicts the mass spectrum of a compound having structure FormulaII-5A.

FIG. 9 depicts the ¹H NMR spectrum of a compound having structureFormula II-5B.

FIG. 10 depicts the mass spectrum of a compound having structure FormulaII-5B.

FIG. 11 depicts the effect of Formulae II-2, II-3, and II-4 againstLeTx-mediated cytotoxicity.

FIG. 12 depicts the ¹H NMR spectrum of the compound of Formula II-8C.

FIG. 13 depicts the ¹H NMR spectrum of the compound of Formula II-13C.

FIG. 14 depicts the ¹H NMR spectrum of the compound of Formula II-18.

FIG. 15 depicts the ¹H NMR spectrum of the compound of Formula II-19.

FIG. 16 depicts the ¹H NMR spectrum of the compound of Formula II-20.

FIG. 17 depicts the ¹H NMR spectrum of the compound of Formula II-21.

FIG. 18 depicts the ¹H NMR spectrum of the compound of Formula II-22.

FIG. 19 depicts the ¹H NMR spectrum of the compound of Formula II-24C.

FIG. 20 depicts the ¹H NMR spectrum of the compound of Formula II-25.

FIG. 21 depicts the ¹H NMR spectrum of the compound of Formula IV-3C inDMSO-d₆.

FIG. 22 depicts the ¹H NMR spectrum of the compound of Formula IV-3C inC₆D₆/DMSO-d₆.

FIG. 23 depicts the ¹H NMR spectrum of the compound of Formula II-26 inDMSO-d₆.

FIG. 24 depicts the computer-generated ORTEP plot of the compound ofFormula II-26.

FIG. 25 depicts the ¹H NMR spectrum of the compound of Formula II-27 inDMSO-d₆.

FIG. 26 depicts the ¹H NMR spectrum of the compound of Formula II-28 inDMSO-d₆.

FIG. 27 depicts the ¹H NMR spectrum of the compound of Formula II-29 inDMSO-d₆.

FIG. 28 depicts the ¹H NMR spectrum of the compound of Formula II-30 inDMSO-d₆.

FIG. 29 depicts the ¹H NMR spectrum of the compound of Formula II-44 inDMSO-d₆.

FIG. 30 depicts the ¹H NMR spectrum of the compound of Formula I-7 inDMSO-d6.

FIG. 31 depicts the ¹H NMR spectrum of the compound of Formula II-47 inDMSO-d6.

FIG. 32 depicts the ¹H NMR spectrum of the compound of Formula II-38 inDMSO-d6.

FIG. 33 depicts the ¹H NMR spectrum of the compound of Formula II-50 inDMSO-d6.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Numerous references are cited herein. The references cited herein,including the U.S. patents cited herein, are each to be consideredincorporated by reference in their entirety into this specification.

Embodiments of the invention include, but are not limited to, providinga method for the preparation of compounds, including novel compounds,for example, including compounds described herein and analogs thereof,and to providing a method for producing pharmaceutically acceptableanti-microbial, anti-cancer, and anti-inflammatory compositions, forexample. The methods can include the compositions in relatively highyield, wherein the compounds and/or their derivatives are among theactive ingredients in these compositions. Other embodiments relate toproviding novel compounds not obtainable by currently available methods.Furthermore, embodiments relate to methods of treating cancer,inflammation, and infectious diseases, particularly those affectinghumans. The methods may include, for example, the step of administeringan effective amount of a member of a class of new compounds. Preferredembodiments relate to the compounds and methods of making and using suchcompounds disclosed herein, but not necessarily in all embodiments ofthe present invention, these objectives are met.

For the compounds described herein, each stereogenic carbon can be of Ror S configuration. Although the specific compounds exemplified in thisapplication can be depicted in a particular configuration, compoundshaving either the opposite stereochemistry at any given chiral center ormixtures thereof are also envisioned. When chiral centers are found inthe derivatives of this invention, it is to be understood that thecompounds encompasses all possible stereoisomers.

Compounds of Formula I

Some embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula I:

In certain embodiments the substituent(s) R₁, R₂, and R₃ separately mayinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted oran unsubstituted variant of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including forexample, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl. Further,in certain embodiments, each of E₁, E₂, E₃ and E₄ can be a heteroatom orsubstituted heteroatoms, for example, a heteroatom separately selectedfrom the group consisting of nitrogen, sulfur and oxygen. The dashedline indicates that the designated bond is either a single bond or adouble bond. In some embodiments, preferably R₁ is a substituted orunsubstituted C₁ to C₅ alkyl. For example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, and pentyl are preferred. Insome embodiments, R₁ is not a substituted or unsubstituted, unbranchedC₆ alkyl.

In some embodiments n can be equal to 1 or equal to 2. When n is equalto 2, the substituents can be the same or can be different. Also, someembodiments include the proviso that R₂ is not cyclohex-2-enyl-carbinolor substituted cyclohex-2-enyl-carbinol. Also, some embodiments includethe proviso that Formula I is not Compound II-16 or Compound II-17.Other embodiments include the proviso that R₂ is notcyclohex-2-enyl-carbinol, when R₃ is methyl. Furthermore, in someembodiments R₃ is not hydrogen.

Preferably, R₂ can be a formyl. For example, the compound may have thefollowing structure I-1:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, the structure of Formula I-1 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, R₂ can be a carbinol. For example, the compound may have thefollowing structure I-2:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

As an example, the structure of Formula I-2 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

As exemplary compound of Formula I can be the compound having thefollowing structure I-3:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The compound of Formula I-3 may have the following stereochemicalstructure:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Another exemplary compound Formula I can be the compound having thefollowing structure I-4:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, the compound of Formula I-4 may have the followingstereochemical structure:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Still a further exemplary compound of Formula I is the compound havingthe following structure I-5:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

For example, the compound of Formula I-5 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

In some embodiments, R₂ of Formula I may be, for example, acyclohex-2-enylidenemethyl. For example, the compound may have thefollowing structure of Formula I-6:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, the compound of Formula I-6 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

In further embodiments, R₂ of Formula I can be, for example, acyclohex-2-enylmethyl. For example, the compound may have the followingstructure of Formula I-7:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, the compound of Formula I-7 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

In other embodiments, R₂ can be a cyclohexylalkylamine.

Also, in other embodiments, R₂ can be a C-Cyclohexyl-methyleneamine. Inothers, R₂ can be a cyclohexanecarbaldehyde O-oxime.

Furthermore, in some embodiments, R₂ can be a cycloalkylacyl.

Compounds of Formula II

Other embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula II:

In certain embodiments the substituent(s) R₁, R₃, and R₄ separately mayinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted oran unsubstituted variant of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters, thiocyano,boronic acids and esters, and halogenated alkyl includingpolyhalogenated alkyl. Further, in certain embodiments, each of E₁, E₂,E₃ and E₄ can be a substituted or unsubstituted heteroatom. For example,the heteroatom can be nitrogen, sulfur and oxygen. The dashed lineindicates that the designated bond is either a single bond or a doublebond.

In some embodiments n or m can be equal to 1, while in others it can beequal to 2. When n or m is equal to 2, the substituents can be the sameor can be different. Also, some embodiments include the proviso thatFormula II is not Compound II-16 or Compound II-17. Further embodimentsinclude the proviso that R₄ is not cyclohex-2-enyl or substitutedcyclohex-2-enyl. Also, some embodiments include the proviso that R₄ isnot cyclohex-2-enyl, when R₃ is methyl. Furthermore, in some embodimentsR₃ is not a hydrogen.

E₅ may be, for example, OH, O, OR₁₀, S, SR₁₁, SO₂R₁₁, NH, NH₂, NOH,NHOH, NR₁₂, and NHOR₁₃, wherein R₁₀₋₁₃ may separately include, forexample, hydrogen, alkyl, substituted alkyl, aryl, heteroaryl and thelike. Also, R₁ can be CH₂CH₂X, wherein X may be, for example, H, F, Cl,Br, and I. R₃ can be methyl. Furthermore, R₄ can be cyclohexyl. Also,each of E₁, E₃ and E₄ can be O and E₂ can be NH. Preferably, R₁ can beCH₂CH₂X, wherein X is selected from the group consisting of H, F, Cl Br,and I; wherein R₄ may include a cyclohexyl; wherein R₃ can be methyl;and wherein each of E₁, E₃ and E₄ can be O and E₂ can be NH.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

For example, an exemplary compound of Formula II has the followingstructure II-1:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Exemplary stereochemistry can be as follows:

In preferred embodiments, the compound of Formula II has any of thefollowing structures II-2, II-3 or II-4, respectively:

It should be noted that the stereochemistry of the above structures canbe changed to the opposite stereochemistry at one or more of the chiralcenters. For example, some embodiments include the following structuresshown without stereochemistry:

In other embodiments wherein R₄ can be 7-oxa-bicyclo[4.1.0]hept-2-yl. Anexemplary compound of Formula II is the following structure II-5:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following are examples of compounds having the structure of FormulaII-5:

In still further embodiments, R₄ may include a substituted or anunsubstituted branched alkyl. For example, a compound of Formula II canbe the following structure II-6:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-6:

As another example, the compound of Formula II can be the followingstructure II-7:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-7:

In other embodiments, R₄ can be a cycloalkyl and E₅ can be an oxygen. Anexemplary compound of Formula II can be the following structure II-8:

R₈ may include, for example, hydrogen (II-8A), fluorine (II-8B),chlorine (II-8C), bromine (II-8D) and iodine (II-8E).

The following is exemplary stereochemistry for a compound having thestructure of Formula II-8:

In some embodiments E5 can be an amine oxide, giving rise to an oxime.An exemplary compound of Formula II has the following structure II-9:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine; R can be hydrogen, alkyl, or substituted alkyl, for example.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-9:

A further exemplary compound of Formula II has the following structureII-10:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-10 (wavy bond indicates that any stereochemistryis allowed):

In some embodiments, E₅ can be NH₂. An exemplary compound of Formula IIhas the following structure II-11:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-11:

In some embodiments, R₄ may include a cycloalkyl and E₅ can be NH₂. Anexemplary compound of Formula II has the following structure II-12:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-12:

A further exemplary compound of Formula II has the following structureII-13:

R₈ may include, for example, hydrogen (II-13A), fluorine (II-13B),chlorine (II-13C), bromine (II-13D) and iodine (II-13E).

The following is exemplary stereochemistry for a compound having thestructure of Formula II-13:

A still further exemplary compound of Formula II has the followingstructure II-14:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-14:

In some embodiments, the compounds of Formula II, may include as R₄ atleast one cycloalkene, for example. Furthermore, in some embodiments,the compounds may include a hydroxy at E₅, for example. A furtherexemplary compound of Formula II has the following structure II-15:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine. In some embodiments, R₈ does not include hydrogen or chlorine.

Exemplary stereochemistry can be as follows:

The following is exemplary stereochemistry for compounds having thestructures II-18 and II-19, respectively:

The compounds of Formulae II-18 and II-19 can be obtained byfermentation, synthesis, or semi-synthesis and isolated/purified as setforth below. Furthermore, the compounds of Formulae II-18 and II-19 canbe used, and are referred to, as “starting materials” to make othercompounds described herein.

In some embodiments, the compounds of Formula II, may include a methylgroup as R₁, for example. A further exemplary compound, Formula II-20,has the following structure and stereochemistry:

In some embodiments, the compounds of Formula II, may includehydroxyethyl as R₁, for example. A further exemplary compound, FormulaII-21, has the following structure and stereochemistry:

In some embodiments, the hydroxyl group of Formula II-21 can beesterified such that R₁ may include ethylpropionate, for example. Anexemplary compound, Formula II-22, has the following structure andstereochemistry:

In some embodiments, the compounds of Formula II may include an ethylgroup as R₃, for example. A further exemplary compound of Formula II hasthe following structure II-23:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine. Exemplary stereochemistry can be as follows:

In some embodiments, the compounds of Formula II-23 may have thefollowing structure and stereochemistry, exemplified by Formula II-24C,where R₃ is ethyl and R₈ is chlorine:

In some embodiments, the compounds of Formula II-15 may have thefollowing stereochemistry, exemplified by the compound of Formula II-25,where R₈ is chlorine:

In some embodiments, the compound of Formula II-15 may have thefollowing stereochemistry, exemplified by the compound of Formula II-26,where R₈ is chlorine:

In some embodiments, the compound of Formula II may have the followingstructure and stereochemistry, exemplified by Formula II-27, where R₁ isethyl:

In some embodiments, the compound of Formula II may have the followingstructure (shown without stereochemistry and with exemplarystereochemistry), exemplified by Formula II-28, where R₁ is methyl:

In some embodiments, the compounds of Formula II may include azidoethylas R₁, for example. A further exemplary compound, Formula II-29, has thefollowing structure and stereochemistry:

In some embodiments, the compounds of Formula II may include propyl asR₁, for example. A further exemplary compound, Formula II-30, has thefollowing structure and stereochemistry:

Still further exemplary compounds, Formulae II-31 and II-32, have thefollowing structure and stereochemistry:

Other exemplary compounds, Formulae II-33, II-34, II-35 and II-36, havethe following structure and stereochemistry:

In some embodiments, the compound of Formula II may include cyanoethylas R₁; for example, the compound of Formula II-37 has the followingstructure and stereochemistry:

In another embodiment, the compound of Formula II may includeethylthiocyanate as R₁; for example, the compound of Formula II-38 hasthe following structure and stereochemistry:

In some embodiments, the compounds of Formula II may include a thiol asR₁, for example. A further exemplary compound, Formula II-39, has thefollowing structure and stereochemistry, where R═H, alkyl, aryl, orsubstituted alkyl or aryl:

In a further exemplary compound, the sulfur of the compound of FormulaII-39 can be oxidized to a sulfoxide (n=1) or sulfone (n=2), forexample, as in the compound of Formula II-40:

In some embodiments, the substituent R₁ of the compound of Formula IImay include a leaving group, for example, a halogen, as in compoundsII-18 or II-19, or another leaving group, such as a sulfonate ester. Oneexample is the methane sulfonate (mesylate) of Formula II-41:

In some embodiments, the substituent R₁ of the compound of Formula IImay include electron acceptors. The electron acceptor may be, forexample, a Lewis acid, such as a boronic acid or ester. An exemplarycompound, Formula II-42, has the following structure andstereochemistry, where n=0, 1, 2, 3, 4, 5, or 6, for example, and whereR═H or alkyl, for example:

Further exemplary compounds of Formula II-42 are the compounds ofFormula II-42A, where n=2 and R═H, and the compound of Formula II-42B,where n=1 and R═H:

In some embodiments where the substituent R₁ of the compound of FormulaII includes an electron acceptor, the electron acceptor may be, forexample, a Michael acceptor. An exemplary compound, Formula II-43 hasthe following structure, where n=0, 1, 2, 3, 4, 5, 6, and where Z is anelectron withdrawing group, for example, CHO, COR, COOR, CONH₂, CN, NO₂,SOR, SO₂R, etc:

A further exemplary compound of Formula II-43 is the compound of FormulaII-43A, where n=1 and Z=CO₂CH₃:

In some embodiments, the compounds of Formula II may include an alkenylgroup as R₁, for example, ethylenyl. A further exemplary compound,Formula II-46, has the following structure and stereochemistry:

Other exemplary compound, Formula II-49 can have the following structureand stereochemistry:

In some embodiments, the compounds can be prodrug esters or thioestersof the compounds of Formula II. For example, the compound of FormulaII-44 (a pro drug thioester of the compound of Formula II-16) has thefollowing structure and stereochemistry:

In another example, the compound of Formula II-47 (a prodrug thioesterof the compound of Formula II-17) has the following structure andstereochemistry:

In yet another example, the compound of Formula II-48 has the followingstructure and stereochemistry:

In another example, the compound of Formula II-50 (prodrug ester of thecompound of Formula II-16) has the following structure andstereochemistry:

Compounds of Formula III

Other embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula III:

In certain embodiments, the substituent(s) R₁ may include, for example,a hydrogen, a halogen, a mono-substituted, a poly-substituted or anunsubstituted variant of the following residues: saturated C₁-C₂₄ alkyl,unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl. Forexample, n can be equal to 1 or 2. When n is equal to 2, thesubstituents can be the same or can be different. The dashed lineindicates that the designated bond is either a single bond or a doublebond.

In certain embodiments, R₄ may be, for example, hydrogen, a halogen, amono-substituted, a poly-substituted or an unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl. For example, m can beequal to 1 or 2. When m is equal to 2, the substituents can be the sameor can be different. Some embodiments include the proviso that FormulaIII is not Compound II-16 or Compound II-17. Further embodiments includethe proviso that R₄ is not cyclohex-2-enyl or substitutedcyclohex-2-enyl. Also, each of E₁, E₂, E₃, E₄ and E₅ may be, forexample, a heteroatom or substituted heteroatom. For example, theheteroatom can be nitrogen, sulfur or oxygen.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

Compounds of Formula IV

Other embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula IV:

In certain embodiments, the substituent(s) R₁, R₃, and R₅ may separatelyinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted oran unsubstituted variants of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, oxy, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano,thio, sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl. Also,each of E₁, E₂, E₃, E₄ and E₅ can be a heteroatom or substitutedheteroatom, for example, nitrogen, sulfur or oxygen. In someembodiments, R₃ is not a hydrogen. n is equal to 1 or 2. When n is equalto 2, the substituents can be the same or can be different. Also, m canbe 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 and if m is more than 1, thenR₅ can be the same or different. Furthermore, the substituents R₅ mayform a ring, for example, an epoxide. The dashed line indicates that thedesignated bond is either a single bond or a double bond.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

In some embodiments, the substituents R₅ may give rise to, for example,a di-substituted cyclohexane. An exemplary compound of Formula IV is thefollowing structure IV-1, with and without exemplary stereochemistry(the wavy bond lines indicate that any stereochemical orientation isallowed):

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine. The substituent(s) R₆ and R₇ may separately include a hydrogen,a halogen, a mono-substituted, a poly-substituted or an unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl. Further, R₆ and R₇both can be the same or different.

For example, an exemplary compound of Formula IV has the followingstructure IV-2:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Exemplary stereochemistry can be as follows:

For example, an exemplary compound of Formula IV has the followingstructure IV-3:

R₈ may include, for example, hydrogen (IV-3A), fluorine (IV-3B),chlorine (IV-3C), bromine (IV-3D) and iodine (IV-3E).

Exemplary stereochemistry can be as follows:

Additional exemplary structure and stereochemistry can be as follows:

For example, an exemplary compound of Formula IV has the followingstructure IV-4:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Exemplary stereochemistry can be as follows:

Compounds of Formula V

Some embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula V:

In certain embodiments, the substituent(s) R₁ and R₅ may separatelyinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted orunsubstituted variants of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, oxy, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano,thio, sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl. Incertain embodiments, each of E₁, E₂, E₃, E₄ and E₅ can be a heteroatomor substituted heteroatom, for example, nitrogen, sulfur or oxygen.Preferably, m may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or11 and if m is more than 1, then R₅ can be the same or different.Furthermore, the substituents R₅ may form a ring, for example, anepoxide. The dashed line indicates that the designated bond is either asingle bond or a double bond.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred. In some embodiments, R₁is not a substituted or unsubstituted, unbranched C₆ alkyl.

Compounds of Formula VI

Some embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula VI:

wherein R₁ can be separately selected from the group consisting of amono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, phenyl, cycloalkylacyl, alkylthio,arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfoneboronic acidesters, and halogenated alkyl including polyhalogenated alkyl. n isequal to 1 or 2, and if n is equal to 2, then R₁ can be the same ordifferent;

wherein R₂, can be selected from the group consisting of hydrogen, ahalogen, mono-substituted, poly-substituted or unsubstituted variants ofthe following residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl (including, for example,cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl;

wherein R₃ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl; whereineach of E₁, E₂, E₃ and E₄ can be a substituted or unsubstitutedheteroatom.

In some embodiments, R₂ is not cyclohex-2-enyl carbinol when one of theR₁ substituents is ethyl or chloroethyl and R₃ is methyl.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred.

wherein R₁₄ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,thioesters, sulfoxide, sulfone, sulfonate esters, thiocyano, andhalogenated alkyl including polyhalogenated alkyl.

In some embodiments, preferably R₁₄ is an alkylthiol or substitutedalkylthiol, and E₃ is an oxygen.

For example, in some embodiments some of the compounds of Formula VI canhave the following structure referred to as Formula VI-1:

wherein R₁ can be separately selected from the group consisting of amono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, phenyl, cycloalkylacyl, alkylthio,arylthio, oxysulfonyl, carboxy, thio, sulfoxide, sulfone, boronic acidesters, and halogenated alkyl including polyhalogenated alkyl. n isequal to 1 or 2, and if n is equal to 2, then R₁ can be the same ordifferent;

wherein R₃ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate esters, thiocyano, boronic acids andesters, and halogenated alkyl including polyhalogenated alkyl; whereineach of E₁, E₂, E₃ and E₄ can be a substituted or unsubstitutedheteroatom.

wherein R₅ can be separately selected from the group consisting of ahydrogen, a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, oxy, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate esters, thiocyano, boronic acids and esters, andhalogenated alkyl including polyhalogenated alkyl, wherein m is 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or 11 and if m is more than 1, then R₅ can bethe same or different; and where the substituents R₅ can form a ring;and wherein each of E₁, E₂, E₃, E₄ and E₅ is a substituted orunsubstituted heteroatom.

In some embodiments, preferably R₁ is a substituted or unsubstituted C₁to C₅ alkyl. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and pentyl are preferred.

wherein R₁₄ can be selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,thioesters, sulfoxide, sulfone, sulfonate esters, thiocyano, andhalogenated alkyl including polyhalogenated alkyl.

For example, the compound has the following structure VI-1A:

Exemplary stereochemistry can be as follows:

For example, an exemplary compound of Formula VI has the followingstructure and stereochemistry VI-1B:

Another example, the compound of Formula VI has the following structureand stereochemistry VI-1C:

Certain embodiments also provide pharmaceutically acceptable salts andpro-drug esters of the compound of Formulae I-VI, and provide methods ofobtaining and purifying such compounds by the methods disclosed herein.

The term “pro-drug ester,” especially when referring to a pro-drug esterof the compound of Formula I synthesized by the methods disclosedherein, refers to a chemical derivative of the compound that is rapidlytransformed in vivo to yield the compound, for example, by hydrolysis inblood or inside tissues. The term “pro-drug ester” refers to derivativesof the compounds disclosed herein formed by the addition of any ofseveral ester- or thioester-forming groups that are hydrolyzed underphysiological conditions. Examples of pro-drug ester groups includepivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl, and methoxymethyl,and thioester, as well as other such groups known in the art, includinga (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other prodrugs can beprepared by preparing a corresponding thioester of the compound, forexample, by reacting with an appropriate thiol, such as thiophenol,Cysteine or derivatives thereof, or propanethiol, for example. Otherexamples of pro-drug ester groups can be found in, for example, T.Higuchi and V. Stella, in “Pro-drugs as Novel Delivery Systems”, Vol.14, A.C.S. Symposium Series, American Chemical Society (1975); and“Bioreversible Carriers in Drug Design: Theory and Application”, editedby E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providingexamples of esters useful as prodrugs for compounds containing carboxylgroups). Each of the above-mentioned references is hereby incorporatedby reference in its entirety.

The term “pro-drug ester,” as used herein, also refers to a chemicalderivative of the compound that is rapidly transformed in vivo to yieldthe compound, for example, by hydrolysis in blood.

The term “pharmaceutically acceptable salt,” as used herein, andparticularly when referring to a pharmaceutically acceptable salt of acompound, including Formulae I-VI, and Formula I-VI as produced andsynthesized by the methods disclosed herein, refers to anypharmaceutically acceptable salts of a compound, and preferably refersto an acid addition salt of a compound. Preferred examples ofpharmaceutically acceptable salt are the alkali metal salts (sodium orpotassium), the alkaline earth metal salts (calcium or magnesium), orammonium salts derived from ammonia or from pharmaceutically acceptableorganic amines, for example C₁-C₇ alkylamine, cyclohexylamine,triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane.With respect to compounds synthesized by the method of this embodimentthat are basic amines, the preferred examples of pharmaceuticallyacceptable salts are acid addition salts of pharmaceutically acceptableinorganic or organic acids, for example, hydrohalic, sulfuric,phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid,for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic,nicotinic, methanesulfonic, p-toluensulfonic or naphthalenesulfonicacid.

Preferred pharmaceutical compositions disclosed herein includepharmaceutically acceptable salts and pro-drug esters of the compound ofFormulae I-VI obtained and purified by the methods disclosed herein.Accordingly, if the manufacture of pharmaceutical formulations involvesintimate mixing of the pharmaceutical excipients and the activeingredient in its salt form, then it is preferred to use pharmaceuticalexcipients which are non-basic, that is, either acidic or neutralexcipients.

It will be also appreciated that the phrase “compounds and compositionscomprising the compound,” or any like phrase, is meant to encompasscompounds in any suitable form for pharmaceutical delivery, as discussedin further detail herein. For example, in certain embodiments, thecompounds or compositions comprising the same may include apharmaceutically acceptable salt of the compound.

In one embodiment the compounds can be used to treat microbial diseases,cancer, and inflammation. Disease is meant to be construed broadly tocover infectious diseases, and also autoimmune diseases, non-infectiousdiseases and chronic conditions. In a preferred embodiment, the diseaseis caused by a microbe, such as a bacterium, a fungi, and protozoa, forexample. The methods of use may also include the steps of administeringa compound or composition comprising the compound to an individual withan infectious disease or cancer. The compound or composition can beadministered in an amount effective to treat the particular infectiousdisease, cancer or inflammatory condition.

The infectious disease may be, for example, one caused by Bacillus, suchas B. anthracis and B. cereus. The infectious disease can be one causedby a protozoa, for example, a Leishmania, a Plasmodium or a Trypanosoma.The compound or composition can be administered with a pharmaceuticallyacceptable carrier, diluent, excipient, and the like.

The cancer may be, for example, a multiple myeloma, a colorectalcarcinoma, a prostate carcinoma, a breast adenocarcinoma, a non-smallcell lung carcinoma, an ovarian carcinoma, a melanoma, and the like.

The inflammatory condition may be, for example, rheumatoid arthritis,asthma, multiple sclerosis, psoriasis, stroke, myocardial infarction,reperfusion injury, and the like.

The term “halogen atom,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,i.e., fluorine, chlorine, bromine, or iodine, with bromine and chlorinebeing preferred.

The term “alkyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, saturated hydrocarbon, with C₁-C₂₄preferred, and C₁-C₆ unbranched or branched, saturated or unsaturated,unsubstituted or substituted hydrocarbons being more preferred, and withmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, andpentyl being most preferred. Among the substituted, saturatedhydrocarbons, C₁-C₂₄ are preferred, with C₁-C₆ mono- and di- andper-halogen substituted saturated hydrocarbons and amino-substitutedhydrocarbons most preferred.

The term “substituted” has its ordinary meaning, as found in numerouscontemporary patents from the related art. See, for example, U.S. Pat.Nos. 6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443;and 6,350,759; all of which are incorporated herein in their entiretiesby reference. Specifically, the definition of substituted is as broad asthat provided in U.S. Pat. No. 6,509,331, which defines the term“substituted alkyl” such that it refers to an alkyl group, preferably offrom 1 to 10 carbon atoms, having from 1 to 5 substituents, andpreferably 1 to 3 substituents, selected from the group consisting ofalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano,halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol,thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Theother above-listed patents also provide standard definitions for theterm “substituted” that are well-understood by those of skill in theart.

The term “cycloalkyl” refers to any non-aromatic hydrocarbon ring,preferably having five to twelve atoms comprising the ring. The term“acyl” refers to alkyl or aryl groups derived from an oxoacid, with anacetyl group being preferred.

The term “alkenyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, unsaturated hydrocarbon includingpolyunsaturated hydrocarbons, with C₁-C₆ unbranched, mono-unsaturatedand di-unsaturated, unsubstituted hydrocarbons being preferred, andmono-unsaturated, di-halogen substituted hydrocarbons being mostpreferred. The term “cycloalkenyl” refers to any non-aromatichydrocarbon ring, preferably having five to twelve atoms comprising thering.

The terms “aryl,” “substituted aryl,” “heteroaryl,” and “substitutedheteroaryl,” as used herein, refer to aromatic hydrocarbon rings,preferably having five, six, or seven atoms, and most preferably havingsix atoms comprising the ring. “Heteroaryl” and “substitutedheteroaryl,” refer to aromatic hydrocarbon rings in which at least oneheteroatom, e.g. oxygen, sulfur, or nitrogen atom, is in the ring alongwith at least one carbon atom. The term “heterocycle” or “heterocyclic”refer to any cyclic compound containing one or more heteroatoms. Thesubstituted aryls, heterocycles and heteroaryls can be substituted withany substituent, including those described above and those known in theart.

The term “alkoxy” refers to any unbranched, or branched, substituted orunsubstituted, saturated or unsaturated ether, with C₁-C₆ unbranched,saturated, unsubstituted ethers being preferred, with methoxy beingpreferred, and also with dimethyl, diethyl, methyl-isobutyl, andmethyl-tert-butyl ethers also being preferred. The term “cycloalkoxy”refers to any non-aromatic hydrocarbon ring, preferably having five totwelve atoms comprising the ring. The term “alkoxy carbonyl” refers toany linear, branched, cyclic, saturated, unsaturated, aliphatic oraromatic alkoxy attached to a carbonyl group. The examples includemethoxycarbonyl group, ethoxycarbonyl group, propyloxycarbonyl group,isopropyloxycarbonyl group, butoxycarbonyl group, sec-butoxycarbonylgroup, tert-butoxycarbonyl group, cyclopentyloxycarbonyl group,cyclohexyloxycarbonyl group, benzyloxycarbonyl group, allyloxycarbonylgroup, phenyloxycarbonyl group, pyridyloxycarbonyl group, and the like.

The terms “pure,” “purified,” “substantially purified,” and “isolated”as used herein refer to the compound of the embodiment being free ofother, dissimilar compounds with which the compound, if found in itsnatural state, would be associated in its natural state. In certainembodiments described as “pure,” “purified,” “substantially purified,”or “isolated” herein, the compound may comprise at least 0.5%, 1%, 5%,10%, or 20%, and most preferably at least 50% or 75% of the mass, byweight, of a given sample.

The terms “derivative,” “variant,” or other similar term refers to acompound that is an analog of the other compound.

Certain of the compounds of Formula I-VI can be obtained and purified orcan be obtained via semi-synthesis from purified compounds as set forthherein. Generally, without being limited thereto, the startingcompounds, such as Compounds II-16, II-17 and II-18), below, can beobtained and the various analogs can be synthesized therefrom. Exemplarynon-limiting syntheses are provided herein.

Production of Starting Compounds I-7, II-16, II-17 and II-18, II-20,II-24C, II-26, II-27 and II-28

The production of starting compounds I-7, II-16, II-17, II-18, II-20,II-24C, II-26, II-27 and II-28 can be carried out by cultivating strainCNB476 and strain NPS21184, a natural variant of strain CNB476, in asuitable nutrient medium under conditions described herein, preferablyunder submerged aerobic conditions, until a substantial amount ofcompounds are detected in the fermentation; harvesting by extracting theactive components from the fermentation broth with a suitable solvent;concentrating the solvent containing the desired components; thensubjecting the concentrated material to chromatographic separation toisolate the compounds from other metabolites also present in thecultivation medium.

The culture (CNB476) was deposited on Jun. 20, 2003 with the AmericanType Culture Collection (ATCC) in Rockville, Md. and assigned the ATCCpatent deposition number PTA-5275. Strain NPS21184, a natural variant ofstrain CNB476, was derived from strain CNB476 as a single colonyisolate. Strain NPS21184 has been deposited to ATCC on Apr. 27, 2005.The ATCC deposit meets all of the requirements of the Budapest treaty.The culture is also maintained at and available from NereusPharmaceutical Culture Collection at 10480 Wateridge Circle, San Diego,Calif. 92121. In addition to the specific microorganism describedherein, it should be understood that mutants, such as those produced bythe use of chemical or physical mutagens including X-rays, etc. andorganisms whose genetic makeup has been modified by molecular biologytechniques, may also be cultivated to produce the starting compoundsI-7, II-16, II-17, II-18, II-20, II-24C, II-26 and II-28.

Fermentation of Strain CNB476 and Strain NPS21184

Production of compounds can be achieved at temperature conducive tosatisfactory growth of the producing organism, e.g. from 16 degree C. to40 degree C., but it is preferable to conduct the fermentation at 22degree C. to 32 degree C. The aqueous medium can be incubated for aperiod of time necessary to complete the production of compounds asmonitored by high pressure liquid chromatography (HPLC), preferably fora period of about 2 to 10 days, on a rotary shaker operating at about 50rpm to 400 rpm, preferably at 150 rpm to 250 rpm, for example. Theproduction of the compounds can also be achieved by cultivating theproduction strain in a bioreactor, such as a fermentor system that issuitable for the growth of the production strain.

Growth of the microorganisms can be achieved by one of ordinary skill ofthe art by the use of appropriate medium. Broadly, the sources of carboninclude glucose, fructose, mannose, maltose, galactose, mannitol andglycerol, other sugars and sugar alcohols, starches and othercarbohydrates, or carbohydrate derivatives such as dextran, cerelose, aswell as complex nutrients such as oat flour, corn meal, millet, corn,and the like. The exact quantity of the carbon source that is utilizedin the medium will depend in part, upon the other ingredients in themedium, but an amount of carbohydrate between 0.5 to 25 percent byweight of the medium can be satisfactorily used, for example. Thesecarbon sources can be used individually or several such carbon sourcescan be combined in the same medium, for example. Certain carbon sourcesare preferred as hereinafter set forth.

The sources of nitrogen include amino acids such as glycine, arginine,threonine, methionine and the like, ammonium salt, as well as complexsources such as yeast extracts, corn steep liquors, distiller solubles,soybean meal, cottonseed meal, fish meal, peptone, and the like. Thevarious sources of nitrogen can be used alone or in combination inamounts ranging from 0.5 to 25 percent by weight of the medium, forexample.

Among the nutrient inorganic salts, which can be incorporated in theculture media, are the customary salts capable of yielding sodium,potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate,and like ions. Also included are trace metals such as cobalt, manganese,iron, molybdenum, zinc, cadmium, and the like.

Pharmaceutical Compositions

In one embodiment, the compounds disclosed herein are used inpharmaceutical compositions. The compounds optionally and preferably areproduced by the methods disclosed herein. The compounds can be used, forexample, in pharmaceutical compositions comprising a pharmaceuticallyacceptable carrier prepared for storage and subsequent administration.Also, embodiments relate to a pharmaceutically effective amount of theproducts and compounds disclosed above in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985), which is incorporated herein by reference in itsentirety. Preservatives, stabilizers, dyes and even flavoring agents canbe provided in the pharmaceutical composition. For example, sodiumbenzoate, ascorbic acid and esters of p-hydroxybenzoic acid can be addedas preservatives. In addition, antioxidants and suspending agents can beused.

The compositions, particularly those of Formulae I-VI, can be formulatedand used as tablets, capsules, or elixirs for oral administration;suppositories for rectal administration; sterile solutions, suspensionsfor injectable administration; patches for transdermal administration,and sub-dermal deposits and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), can be utilized.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or other organic oilssuch as soybean, grapefruit or almond oils, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions can be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions can be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. Such formulations can be madeusing methods known in the art (see, for example, U.S. Pat. No.5,733,888 (injectable compositions); U.S. Pat. No. 5,726,181 (poorlywater soluble compounds); U.S. Pat. No. 5,707,641 (therapeuticallyactive proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilicagents); U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S.Pat. No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676(particulate medicaments); U.S. Pat. No. 5,654,286 (topicalformulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S. Pat. No.5,445,829 (extended release formulations); U.S. Pat. No. 5,653,987(liquid formulations); U.S. Pat. No. 5,641,515 (controlled releaseformulations) and U.S. Pat. No. 5,601,845 (spheroid formulations); allof which are incorporated herein by reference in their entireties.

Further disclosed herein are various pharmaceutical compositions wellknown in the pharmaceutical art for uses that include topical,intraocular, intranasal, and intraauricular delivery. Pharmaceuticalformulations include aqueous ophthalmic solutions of the activecompounds in water-soluble form, such as eyedrops, or in gellan gum(Shedden et al., Clin. Ther., 23(3):440-50 (2001)) or hydrogels (Mayeret al., Ophthalmologica, 210(2):101-3 (1996)); ophthalmic ointments;ophthalmic suspensions, such as microparticulates, drug-containing smallpolymeric particles that are suspended in a liquid carrier medium(Joshi, A. 1994 J Ocul Pharmacol 10:29-45), lipid-soluble formulations(Alm et al., Prog. Clin. Biol. Res., 312:447-58 (1989)), andmicrospheres (Mordenti, Toxicol. Sci., 52(1):101-6 (1999)); and ocularinserts. All of the above-mentioned references, are incorporated hereinby reference in their entireties. Such suitable pharmaceuticalformulations are most often and preferably formulated to be sterile,isotonic and buffered for stability and comfort. Pharmaceuticalcompositions may also include drops and sprays often prepared tosimulate in many respects nasal secretions to ensure maintenance ofnormal ciliary action. As disclosed in Remington's PharmaceuticalSciences (Mack Publishing, 18^(th) Edition), which is incorporatedherein by reference in its entirety, and well-known to those skilled inthe art, suitable formulations are most often and preferably isotonic,slightly buffered to maintain a pH of 5.5 to 6.5, and most often andpreferably include anti-microbial preservatives and appropriate drugstabilizers. Pharmaceutical formulations for intraauricular deliveryinclude suspensions and ointments for topical application in the ear.Common solvents for such aural formulations include glycerin and water.

When used as an anti-cancer, anti-inflammatory or anti-microbialcompound, for example, the compounds of Formulae I-VI or compositionsincluding Formulae I-VI can be administered by either oral or non-oralpathways. When administered orally, it can be administered in capsule,tablet, granule, spray, syrup, or other such form. When administerednon-orally, it can be administered as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like, when administered via injection, subcutaneously,intraperitoneally, intravenously, intramuscularly, or the like.

In one embodiment, the anti-cancer, anti-inflammatory or anti-microbialcan be mixed with additional substances to enhance their effectiveness.In one embodiment, the anti-microbial is combined with an additionalanti-microbial. In another embodiment, the anti-microbial is combinedwith a drug or medicament that is helpful to a patient that is takinganti-microbials.

The compounds can be administered or used in combination with treatmentssuch as chemotherapy, radiation, and biologic therapies. In someembodiments the compounds can be administered or used with achemotherapeutic agent. Examples of such chemotherapeutics includeAlkaloids, alkylating agents, antibiotics, antimetabolites, enzymes,hormones, platinum compounds, immunotherapeutics (antibodies, T-cells,epitopes), biological response modifiers (BRMs), and the like. Examplesinclude, Vincristine, Vinblastine, Vindesine, Paclitaxel (Taxol),Docetaxel, topoisomerase inhibitors epipodophyllotoxins (Etoposide(VP-16), Teniposide (VM-26)), Camptothecin, nitrogen mustards(cyclophosphamide), Nitrosoureas, Carmustine, lomustine, dacarbazine,hydroxymethylmelamine, thiotepa and mitocycin C, Dactinomycin(Actinomycin D), anthracycline antibiotics (Daunorubicin, Daunomycin,Cerubidine), Doxorubicin (Adriamycin), Idarubicin (Idamycin),Anthracenediones (Mitoxantrone), Bleomycin (Blenoxane), Plicamycin(Mithramycin, Antifolates (Methotrexate (Folex, Mexate)), purineantimetabolites (6-mercaptopurine (6-MP, Purinethol) and 6-thioguanine(6-TG). The two major anticancer drugs in this category are6-mercaptopurine and 6-thioguanine, Chlorodeoxyadenosine andPentostatin, Pentostatin (2′-deoxycoformycin), pyrimidine antagonists,fluoropyrimidines (5-fluorouracil(Adrucil), 5-fluorodeoxyuridine (FdUrd)(Floxuridine)), Cytosine Arabinoside (Cytosar, ara-C), Fludarabine,L-ASPARAGINASE, Hydroxyurea, glucocorticoids, antiestrogens, tamoxifen,nonsteroidal antiandrogens, flutamide, aromatase inhibitorsAnastrozole(Arimidex), Cisplatin, 6-Mercaptopurine and Thioguanine,Methotrexate, Cytoxan, Cytarabine, L-Asparaginase, Steroids: Prednisoneand Dexamethasone. Also, proteasome inhibitors such as bortezomib can beused in combination with the instant compounds, for example. Examples ofbiologics can include agents such as TRAIL antibodies to TRAIL,integrins such as alpha-V-beta-3 (αVβ3) and/or other cytokine/growthfactors that are involved in angiogenesis, VEGF, EGF, FGF and PDGF. Insome aspects, the compounds can be conjugated to or delivered with anantibody. The above-described combination methods can be used to treat avariety of conditions, including cancer and neoplastic diseases,inflammation, and microbial infections.

Methods of Administration

In an alternative embodiment, the disclosed chemical compounds and thedisclosed pharmaceutical compositions are administered by a particularmethod as an anti-microbial. Such methods include, among others, (a)administration though oral pathways, which administration includesadministration in capsule, tablet, granule, spray, syrup, or other suchforms; (b) administration through non-oral pathways, whichadministration includes administration as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like; administration via injection, subcutaneously,intraperitoneally, intravenously, intramuscularly, intradermally, or thelike; as well as (c) administration topically, (d) administrationrectally, or (e) administration vaginally, as deemed appropriate bythose of skill in the art for bringing the compound of the presentembodiment into contact with living tissue; and (f) administration viacontrolled released formulations, depot formulations, and infusion pumpdelivery. As further examples of such modes of administration and asfurther disclosure of modes of administration, disclosed herein arevarious methods for administration of the disclosed chemical compoundsand pharmaceutical compositions including modes of administrationthrough intraocular, intranasal, and intraauricular pathways.

The pharmaceutically effective amount of the compositions that includethe described compounds, including those of Formulae I-VI, required as adose will depend on the route of administration, the type of animal,including human, being treated, and the physical characteristics of thespecific animal under consideration. The dose can be tailored to achievea desired effect, but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize.

In practicing the methods of the embodiment, the products orcompositions can be used alone or in combination with one another, or incombination with other therapeutic or diagnostic agents. These productscan be utilized in vivo ordinarily in a mammal, preferably in a human,or in vitro. In employing them in vivo the products or compositions canbe administered to the mammal in a variety of ways, includingparenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, vaginally, nasally or intraperitoneally,employing a variety of dosage forms. Such methods may also be applied totesting chemical activity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired affects and thetherapeutic indication. Typically, dosages can be between about 10microgram/kg and 100 mg/kg body weight, preferably between about 100microgram/kg and 10 mg/kg body weight. Alternatively dosages can bebased and calculated upon the surface area of the patient, as understoodby those of skill in the art. Administration is preferably oral on adaily or twice daily basis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. See forexample, Fingl et al., in The Pharmacological Basis of Therapeutics,1975, which is incorporated herein by reference in its entirety. Itshould be noted that the attending physician would know how to and whento terminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions. Conversely, the attending physician would also knowto adjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administrated dosein the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above can be used in veterinary medicine.

Depending on the specific conditions being treated, such agents can beformulated and administered systemically or locally. A variety oftechniques for formulation and administration can be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990), which is incorporated herein by reference in itsentirety. Suitable administration routes may include oral, rectal,transdermal, vaginal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

For injection, the agents of the embodiment can be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theembodiment into dosages suitable for systemic administration is withinthe scope of the embodiment. With proper choice of carrier and suitablemanufacturing practice, the compositions disclosed herein, inparticular, those formulated as solutions, can be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the embodiment to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly can be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents can be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules canbe directly administered intracellularly.

Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. The preparations formulated for oraladministration can be in the form of tablets, dragees, capsules, orsolutions. The pharmaceutical compositions can be manufactured in amanner that is itself known, for example, by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, can be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, dogs or monkeys, can be determined using known methods. Theefficacy of a particular compound can be established using several artrecognized methods, such as in vitro methods, animal models, or humanclinical trials. Art-recognized in vitro models exist for nearly everyclass of condition, including the conditions abated by the compoundsdisclosed herein, including cancer, cardiovascular disease, and variousimmune dysfunction, and infectious diseases. Similarly, acceptableanimal models can be used to establish efficacy of chemicals to treatsuch conditions. When selecting a model to determine efficacy, theskilled artisan can be guided by the state of the art to choose anappropriate model, dose, and route of administration, and regime. Ofcourse, human clinical trials can also be used to determine the efficacyof a compound in humans.

When used as an anti-microbial, anti-cancer, or anti-inflammatory agent,the compounds disclosed herein can be administered by either oral or anon-oral pathways. When administered orally, it can be administered incapsule, tablet, granule, spray, syrup, or other such form. Whenadministered non-orally, it can be administered as an aqueoussuspension, an oily preparation or the like or as a drip, suppository,salve, ointment or the like, when administered via injection,subcutaneously, intraperitoneally, intravenously, intramuscularly,intradermally, or the like. Controlled release formulations, depotformulations, and infusion pump delivery are similarly contemplated.

The compositions disclosed herein in pharmaceutical compositions mayalso comprise a pharmaceutically acceptable carrier. Such compositionscan be prepared for storage and for subsequent administration.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).For example, such compositions can be formulated and used as tablets,capsules or solutions for oral administration; suppositories for rectalor vaginal administration; sterile solutions or suspensions forinjectable administration. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients include, but are not limited to, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), can be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize.

The products or compositions of the embodiment, as described above, canbe used alone or in combination with one another, or in combination withother therapeutic or diagnostic agents. These products can be utilizedin vivo or in vitro. The useful dosages and the most useful modes ofadministration will vary depending upon the age, weight and animaltreated, the particular compounds employed, and the specific use forwhich these composition or compositions are employed. The magnitude of adose in the management or treatment for a particular disorder will varywith the severity of the condition to be treated and to the route ofadministration, and depending on the disease conditions and theirseverity, the compositions can be formulated and administered eithersystemically or locally. A variety of techniques for formulation andadministration can be found in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Co., Easton, Pa. (1990).

To formulate the compounds of Formulae I-VI as an anti-microbial, ananti-cancer, or an anti-inflammatory agent, known surface active agents,excipients, smoothing agents, suspension agents and pharmaceuticallyacceptable film-forming substances and coating assistants, and the likecan be used. Preferably alcohols, esters, sulfated aliphatic alcohols,and the like can be used as surface active agents; sucrose, glucose,lactose, starch, crystallized cellulose, mannitol, light anhydroussilicate, magnesium aluminate, magnesium methasilicate aluminate,synthetic aluminum silicate, calcium carbonate, sodium acid carbonate,calcium hydrogen phosphate, calcium carboxymethyl cellulose, and thelike can be used as excipients; magnesium stearate, talc, hardened oiland the like can be used as smoothing agents; coconut oil, olive oil,sesame oil, peanut oil, soya can be used as suspension agents orlubricants; cellulose acetate phthalate as a derivative of acarbohydrate such as cellulose or sugar, or methylacetate-methacrylatecopolymer as a derivative of polyvinyl can be used as suspension agents;and plasticizers such as ester phthalates and the like can be used assuspension agents. In addition to the foregoing preferred ingredients,sweeteners, fragrances, colorants, preservatives and the like can beadded to the administered formulation of the compound produced by themethod of the embodiment, particularly when the compound is to beadministered orally.

The compounds and compositions can be orally or non-orally administeredto a human patient in the amount of about 0.001 mg/kg/day to about10,000 mg/kg/day of the active ingredient, and more preferably about 0.1mg/kg/day to about 100 mg/kg/day of the active ingredient at,preferably, one time per day or, less preferably, over two to about tentimes per day. Alternatively and also preferably, the compound producedby the method of the embodiment may preferably be administered in thestated amounts continuously by, for example, an intravenous drip. Thus,for the example of a patient weighing 70 kilograms, the preferred dailydose of the active or anti-infective ingredient would be about 0.07mg/day to about 700 gm/day, and more preferable, 7 mg/day to about 7grams/day. Nonetheless, as will be understood by those of skill in theart, in certain situations it can be necessary to administer theanti-cancer, anti-inflammatory or the anti-infective compound of theembodiment in amounts that excess, or even far exceed, the above-stated,preferred dosage range to effectively and aggressively treatparticularly advanced cancers or infections.

In the case of using the anti-cancer, anti-inflammatory, oranti-microbial produced by methods of the embodiment as a biochemicaltest reagent, the compound produced by methods of the embodimentinhibits the progression of the disease when it is dissolved in anorganic solvent or hydrous organic solvent and it is directly applied toany of various cultured cell systems. Usable organic solvents include,for example, methanol, methylsulfoxide, and the like. The formulationcan, for example, be a powder, granular or other solid inhibitor, or aliquid inhibitor prepared using an organic solvent or a hydrous organicsolvent. While a preferred concentration of the compound produced by themethod of the embodiment for use as an anti-microbial, anticancer oranti-tumor compound is generally in the range of about 1 to about 100μg/ml, the most appropriate use amount varies depending on the type ofcultured cell system and the purpose of use, as will be appreciated bypersons of ordinary skill in the art. Also, in certain applications itcan be necessary or preferred to persons of ordinary skill in the art touse an amount outside the foregoing range.

In one embodiment, the method of using a compound as an anti-microbial,anti-cancer or anti-inflammatory involves administering an effectiveamount of any of the compounds of Formulae I-VI or compositions of thosecompounds. In a preferred embodiment, the method involves administeringthe compound represented by Formula II, to a patient in need of ananti-microbial, until the need is effectively reduced or more preferablyremoved.

As will be understood by one of skill in the art, “need” is not anabsolute term and merely implies that the patient can benefit from thetreatment of the anti-microbial, the anti-cancer, or anti-inflammatoryin use. By “patient” what is meant is an organism that can benefit bythe use of an anti-microbial, anti-cancer or anti-inflammatory agent.For example, any organism with B. anthracis, Plasmodium, Leishmania,Trypanosoma, and the like, may benefit from the application of ananti-microbial that may in turn reduce the amount of microbes present inthe patient. As another example, any organism with cancer, such as, acolorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, anon-small cell lung carcinoma, an ovarian carcinoma, multiple myelomas,a melanoma, and the like, may benefit from the application of ananti-cancer agent that may in turn reduce the amount of cancer presentin the patient. Furthermore, any organism with an inflammatoryconditions, such as, rheumatoid arthritis, asthma, multiple sclerosis,psoriasis, stroke, reperfusion injury, myocardial infarction, and thelike, may benefit from the application of an anti-inflammatory that mayin turn reduce the amount of cells associated with the inflammatoryresponse present in the patient. In one embodiment, the patient's healthmay not require that an anti-microbial, anti-cancer, oranti-inflammatory be administered, however, the patient may still obtainsome benefit by the reduction of the level of microbes, cancer cells, orinflammatory cells present in the patient, and thus be in need. In oneembodiment, the anti-microbial or anti-cancer agent is effective againstone type of microbe or cancer, but not against other types; thus,allowing a high degree of selectivity in the treatment of the patient.In other embodiments, the anti-inflammatory can be effective againstinflammatory conditions characterized by different cells associated withthe inflammation. In choosing such an anti-microbial, anti-cancer oranti-inflammatory agent, the methods and results disclosed in theExamples can be useful. In an alternative embodiment, the anti-microbialcan be effective against a broad spectrum of microbes, preferably abroad spectrum of foreign, and, more preferably, harmful bacteria, tothe host organism. In embodiments, the anti-cancer and/oranti-inflammatory agent can be effective against a broad spectrum ofcancers and inflammatory conditions/cells/substances. In yet anotherembodiment, the anti-microbial is effective against all microbes, eventhose native to the host. Examples of microbes that can be targets ofanti-microbials, include, but are not limited to, B. anthracis,Plasmodium, Leishmania, Trypanosoma, and the like. In still furtherembodiments, the anti-cancer agent is effective against a broad spectrumof cancers or all cancers. Examples of cancers, against which thecompounds can be effective include a colorectal carcinoma, a prostatecarcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, anovarian carcinoma, multiple myelomas, a melanoma, and the like.Exemplary inflammatory conditions against which the agents are effectiveinclude rheumatoid arthritis, asthma, multiple sclerosis, psoriasis,stroke, myocardial infarction, and the like.

“Therapeutically effective amount,” “pharmaceutically effective amount,”or similar term, means that amount of drug or pharmaceutical agent thatwill result in a biological or medical response of a cell, tissue,system, animal, or human that is being sought. In a preferredembodiment, the medical response is one sought by a researcher,veterinarian, medical doctor, or other clinician.

“Anti-microbial” refers to a compound that reduces the likelihood ofsurvival of microbes, or blocks or alleviates the deleterious effects ofa microbe. In one embodiment, the likelihood of survival is determinedas a function of an individual microbe; thus, the anti-microbial willincrease the chance that an individual microbe will die. In oneembodiment, the likelihood of survival is determined as a function of apopulation of microbes; thus, the anti-microbial will increase thechances that there will be a decrease in the population of microbes. Inone embodiment, anti-microbial means antibiotic or other similar term.Such anti-microbials are capable of blocking the harmful effects,destroying or suppressing the growth or reproduction of microorganisms,such as bacteria. For example, such antibacterials and otheranti-microbials are described in Antibiotics, Chemotherapeutics andAntibacterial Agents for Disease Control (M. Grayson, editor, 1982), andE. Gale et al., The Molecular Basis of Antibiotic Action 2d edition(1981). In another embodiment, an anti-microbial will not change thelikelihood of survival, but will change the chances that the microbeswill be harmful to the host in some way. For instance, if the microbesecretes a substance that is harmful to the host, the anti-microbial mayact upon the microbe to stop the secretion or may counteract or blockthe harmful effect. In one embodiment, an anti-microbial, while,increasing the likelihood that the microbe(s) will die, is minimallyharmful to the surrounding, non-microbial, cells. In an alternativeembodiment, it is not important how harmful the anti-microbial is tosurrounding, nonmicrobial, cells, as long as it reduces the likelihoodof survival of the microbe.

“Anti-cancer agent” refers to a compound or composition including thecompound that reduces the likelihood of survival of a cancer cell. Inone embodiment, the likelihood of survival is determined as a functionof an individual cancer cell; thus, the anti-cancer agent will increasethe chance that an individual cancer cell will die. In one embodiment,the likelihood of survival is determined as a function of a populationof cancer cells; thus, the anti-cancer agent will increase the chancesthat there will be a decrease in the population of cancer cells. In oneembodiment, anti-cancer agent means chemotherapeutic agent or othersimilar term.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of a neoplastic disease, such as cancer. Examples ofchemotherapeutic agents include alkylating agents, such as a nitrogenmustard, an ethyleneimine and a methylmelamine, an alkyl sulfonate, anitrosourea, and a triazene, folic acid antagonists, anti-metabolites ofnucleic acid metabolism, antibiotics, pyrimidine analogs,5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids,triazol nucleosides, corticosteroids, a natural product such as a vincaalkaloid, an epipodophyllotoxin, an antibiotic, an enzyme, a taxane, anda biological response modifier or antibodies to biological responsemodifiers or other agents; miscellaneous agents such as a platinumcoordination complex, an anthracenedione, an anthracycline, asubstituted urea, a methyl hydrazine derivative, or an adrenocorticalsuppressant; or a hormone or an antagonist such as anadrenocorticosteroid, a progestin, an estrogen, an antiestrogen, anandrogen, an antiandrogen, or a gouadotropin-releasing hormone analog.Specific examples include Adriamycin, Doxorubicin, 5-Fluorouracil,Cytosine arabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan,Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan,Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,Mitoxantrone, Vincristine, Vinorelbine, Carboplatin, Teniposide,Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,Esperamicins, Melphalan, and other related nitrogen mustards. Alsoincluded in this definition are hormonal agents that act to regulate orinhibit hormone action on tumors, such as tamoxifen and onapristone.

The anti-cancer agent may act directly upon a cancer cell to kill thecell, induce death of the cell, to prevent division of the cell, and thelike. Alternatively, the anti-cancer agent may indirectly act upon thecancer cell by limiting nutrient or blood supply to the cell, forexample. Such anti-cancer agents are capable of destroying orsuppressing the growth or reproduction of cancer cells, such as acolorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, anon-small cell lung carcinoma, an ovarian carcinoma, multiple myelomas,a melanoma, and the like.

A “neoplastic disease” or a “neoplasm” refers to a cell or a populationof cells, including a tumor or tissue (including cell suspensions suchas bone marrow and fluids such as blood or serum), that exhibitsabnormal growth by cellular proliferation greater than normal tissue.Neoplasms can be benign or malignant.

An “inflammatory condition” includes, for example, conditions such asischemia, septic shock, autoimmune diseases, rheumatoid arthritis,inflammatory bowel disease, systemic lupus eythematosus, multiplesclerosis, asthma, osteoarthritis, osteoporosis, fibrotic diseases,dermatosis, including psoriasis, atopic dermatitis and ultravioletradiation (UV)-induced skin damage, psoriatic arthritis, alkylosingspondylitis, tissue and organ rejection, Alzheimer's disease, stroke,atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer,Hodgkins disease, cachexia, inflammation associated with infection andcertain viral infections, including acquired immune deficiency syndrome(AIDS), adult respiratory distress syndrome and Ataxia Telangiestasia.

In one embodiment, a described compound, preferably a compound havingthe Formulae I-VI, including those as described herein, is considered aneffective anti-microbial, anti-cancer, or anti-inflammatory if thecompound can influence 10% of the microbes, cancer cells, orinflammatory cells, for example. In a more preferred embodiment, thecompound is effective if it can influence 10 to 50% of the microbes,cancer cells, or inflammatory cells. In an even more preferredembodiment, the compound is effective if it can influence 50-80% of themicrobes, cancer cells, or inflammatory cells. In an even more preferredembodiment, the compound is effective if it can influence 80-95% of themicrobes, cancer cells, or inflammatory cells. In an even more preferredembodiment, the compound is effective if it can influence 95-99% of themicrobes, cancer cells, or inflammatory cells. “Influence” is defined bythe mechanism of action for each compound. Thus, for example, if acompound prevents the reproduction of microbes, then influence is ameasure of prevention of reproduction. Likewise, if a compound destroysmicrobes, then influence is a measure of microbe death. Also, forexample, if a compound prevents the division of cancer cells, theninfluence is a measure of prevention of cancer cell division. Further,for example, if a compound prevents the proliferation of inflammatorycells, then influence is a measure of prevention of inflammatory cellproliferation. Not all mechanisms of action need be at the samepercentage of effectiveness. In an alternative embodiment, a lowpercentage effectiveness can be desirable if the lower degree ofeffectiveness is offset by other factors, such as the specificity of thecompound, for example. Thus a compound that is only 10% effective, forexample, but displays little in the way of harmful side-effects to thehost, or non-harmful microbes or cells, can still be consideredeffective.

In one embodiment, the compounds described herein are administeredsimply to remove microbes, cancer cells or inflammatory cells, and neednot be administered to a patient. For example, in situations wheremicrobes can present a problem, such as in food products, the compoundsdescribed herein can be administered directly to the products to reducethe risk of microbes in the products. Alternatively, the compounds canbe used to reduce the level of microbes present in the surroundingenvironment, such working surfaces. As another example, the compoundscan be administered ex vivo to a cell sample, such as a bone marrow orstem cell transplant to ensure that only non-cancerous cells areintroduced into the recipient. After the compounds are administered theymay optionally be removed. This can be particularly desirable insituations where work surfaces or food products may come into contactwith other surfaces or organisms that could risk being harmed by thecompounds. In an alternative embodiment, the compounds can be left inthe food products or on the work surfaces to allow for a moreprotection. Whether or not this is an option will depend upon therelative needs of the situation and the risks associated with thecompound, which in part can be determined as described in the Examplesbelow.

The following non-limiting examples are meant to describe the preferredembodiments of the methods. Variations in the details of the particularmethods employed and in the precise chemical compositions obtained willundoubtedly be appreciated by those of skill in the art.

EXAMPLES Example 1 Fermentation of Starting Compound II-16 and Compoundsof Formulae I-7, II-17, II-20, and II-24C, II-26 and II-28 using StrainCNB476

Strain CNB476 was grown in a 500-ml flask containing 100 ml ofvegetative medium consisting of the following per liter of deionizedwater: glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone, 5 g; andsynthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The firstseed culture was incubated at 28 degree C. for 3 days on a rotary shakeroperating at 250 rpm. Five ml each of the first seed culture wasinoculated into three 500-ml flasks containing of 100 ml of thevegetative medium. The second seed cultures were incubated at 28 degreeC. and 250 rpm on a rotary shaker for 2 days. Five ml each of the secondseed culture was inoculated into thirty-five 500-ml flasks containing of100 ml of the vegetative medium. The third seed cultures were incubatedat 28 degree and 250 rpm on a rotary shaker for 2 days. Five ml each ofthe third seed culture was inoculated into four hundred 500-ml flaskscontaining 100 ml of the Production Medium A consisting of the followingper liter of deionized water: starch, 10 g; yeast extract, 4 g; Hy-Soy,4 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calciumcarbonate, 1 g; and synthetic sea salt (Instant Ocean, AquariumSystems), 30 g. The production cultures were incubated at 28 degree C.and 250 rpm on rotary shakers for 1 day. Approximately 2 to 3 grams ofsterile Amberlite XAD-7 resin were added to the production cultures. Theproduction cultures were further incubated at 28 degree C. and 250 rpmon rotary shakers for 5 days and achieved a titer of Compound II-16about 200 mg/L. The culture broth was filtered through cheese cloth torecover the Amberlite XAD-7 resin. The resin was extracted with 2 times6 liters ethyl acetate followed by 1 time 1.5 liters ethyl acetate. Thecombined extracts were dried in vacuo. The dried extract was thenprocessed for the recovery of Compound II-16 and the compounds ofFormulae I-7, II-20, II-24C, II-26 and II-28.

Example 2 Fermentation of Starting Compound II-16 and compounds ofFormulae I-7, II-17, II-20, II-24C, II-26 and II-28 using StrainNPS21184

Strain NPS21184 was grown in a 500-ml flask containing 100 ml ofvegetative medium consisting of the following per liter of deionizedwater: glucose, 8 g; yeast extract, 6 g; Hy-Soy, 6 g; and synthetic seasalt (Instant Ocean, Aquarium Systems), 30 g. The first seed culture wasincubated at 28 degree C. for 3 days on a rotary shaker operating at 250rpm. Five ml of the first seed culture was inoculated into 500-ml flaskcontaining of 100 ml of the vegetative medium. The second seed cultureswere incubated at 28 degree C. and 250 rpm on a rotary shaker for 2days. Five ml each of the second seed culture was inoculated into 500-mlflask containing of 100 ml of the vegetative medium. The third seedcultures were incubated at 28 degree and 250 rpm on a rotary shaker for2 days. Five ml each of the third seed culture was inoculated into500-ml flask containing 100 ml of the Production Medium B consisting ofthe following per liter of deionized water: starch, 20 g; yeast extract,4 g; Hy-Soy, 8 g; ferric sulfate, 40 mg; potassium bromide, 100 mg;calcium carbonate, 1 g; and synthetic sea salt (Instant Ocean, AquariumSystems), 30 g. The production cultures were incubated at 28 degree C.and 250 rpm on rotary shakers for 1 day. Approximately 2 to 3 grams ofsterile Amberlite XAD-7 resin were added to the production culture. Theproduction culture was further incubated at 28 degree C. and 250 rpm onrotary shaker for 4 days and achieved a titer of 350-400 mg/L forCompound II-16.

Alternatively, the production of the compounds can be achieved in a 42 Lfermentor system using strain NPS21184. Strain NPS21184 was grown in a500-ml flask containing 100 ml of vegetative medium consisting of thefollowing per liter of deionized water: glucose, 8 g; yeast extract, 6g; Hy-Soy, 6 g; and synthetic sea salt (Instant Ocean, AquariumSystems), 30 g. The first seed culture was incubated at 28 degree C. for3 days on a rotary shaker operating at 250 rpm. Five ml of the firstseed culture was inoculated into 500-ml flask containing of 100 ml ofthe vegetative medium. The second seed cultures were incubated at 28degree C. and 250 rpm on a rotary shaker for 2 days. Twenty ml each ofthe second seed culture was inoculated into 2.8 L Fernbach flaskcontaining of 400 ml of the vegetative medium. The third seed cultureswere incubated at 28 degree and 250 rpm on a rotary shaker for 2 days.1.2 L of the third seed culture was inoculated into a 42 L fermentorcontaining 26 L of Production Medium A. Production Medium B andProduction Medium C, with the following composition, can also be used.Production Medium C consisting of the following per liter of deionizedwater: starch, 15 g; yeast extract 6 g; Hy-Soy, 6 g; ferric sulfate, 40mg; potassium bromide, 100 mg; calcium carbonate, 1 g; and synthetic seasalt (Instant Ocean, Aquarium Systems), 30 g. The fermentor cultureswere operated at the following parameters: temperature, 28 degree C.;agitation, 200 rpm; aeration, 13 L/min and back pressure, 4.5 psi. At 36to 44 hours of the production cycle, approximately 600 grams of sterileAmberlite XAD-7 resin were added to the fermentor culture. Theproduction culture was further incubated at the above operatingparameters until day 4 of the production cycle. The aeration rate waslowered to 8 L/min. At day 5 of the production cycle, the fermentorculture achieved a titer of about 300 mg/L for Compound II-16. Theculture broth was filtered through cheese cloth to recover the AmberliteXAD-7 resin. The resin was extracted with 2 times 4.5 L liters ethylacetate followed by 1 time 1.5 liters ethyl acetate. The combinedextracts were dried in vacuo. The dried extract was then processed forthe recovery of Compound II-16 and the compounds of Formulae I-7, II-17,II-20, II-24C, II-26 and II-28.

Example 3 Purification of Starting Compound II-16 and Compounds ofFormulae II-20, II-24C, II-26 and II-28 3A: Purification of II-16,II-20, II-24C, II-26 and II-28

The pure Compound II-16 and compounds of Formulae II-20, II-24C, II-26and II-28 were obtained by flash chromatography followed by HPLC. Eightgrams crude extract containing 3.8 grams Compound II-16 and lesserquantities of II-20, II-24C, II-26 and II-28 was processed by flashchromatography using Biotage Flash40i system and Flash 40M cartridge(KP-Sil Silica, 32-63 μm, 90 grams). The flash chromatography wasdeveloped by the following step gradient:

1. Hexane (1 L)

2. 10% Ethyl acetate in hexane (1 L)

3. 20% Ethyl acetate in hexane, first elution (1 L)

4. 20% Ethyl acetate in hexane, second elution (1 L)

5. 20% Ethyl acetate in hexane, third elution (1 L)

6. 25% Ethyl acetate in hexane (1 L)

7. 50% Ethyl acetate in hexane (1 L)

8. Ethyl acetate (1 L)

Fractions containing Compound II-16 in greater or equal to 70% UV purityby HPLC were pooled and subject to HPLC purification, as describedbelow, to obtain Compound II-16, along with II-20 and II-24C, each aspure compounds

Column Phenomenex Luna 10 u Silica Dimensions 25 cm × 21.2 mm ID Flowrate 25 ml/min Detection ELSD Solvent Gradient of 24% EtOAc/hexane for19 min, 24% EtOAc/hexane to 100% EtOAc in 1 min, then 100% EtOAc for 4min

The fraction enriched in Compound II-16 (described above; ˜70% pure withrespect to Compound II-16) was dissolved in acetone (60 mg/ml). Aliquots(950 ul) of this solution were injected onto a normal-phase HPLC columnusing the conditions described above. Compound II-16 typically elutedafter 14 minutes and compounds II-24C and II-26 co-eluted as a singlepeak at 11 min. When parent samples containing compounds II-17, II-20and II-28 were processed, Compound II-17 eluted at 22 minutes, whileII-20 and II-28 co-eluted at 23 minutes during the 100% ethyl acetatewash. Fractions containing Compound II-16 and minor analogs were pooledbased on composition of compounds present, and evaporated under reducedpressure on a rotary evaporator. This process yielded pure CompoundII-16, as well as separate fractions containing minor compounds II-20,II-24C, II-26 and II-28, which were further purified as described below.

Sample containing II-24C and II-26 generated from the process describedabove were further separated using reversed-phase preparative HPLC asfollows. The sample (70 mg) was dissolved in acetonitrile at aconcentration of 10 mg/ml, and 500 μl was loaded on an HPLC column ofdimensions 21 mm i.d. by 15 cm length containing Eclipse XDB-C18support. The solvent gradient increased linearly from 15%acetonitrile/85% water to 100% acetonitrile over 23 minutes at a flowrate of 14.5 ml/min. The solvent composition was held at 100%acetonitrile for 3 minutes before returning to the starting solventmixture. Compound II-26 eluted at 17.5 minutes while compound II-24Celuted at 19 minutes under these conditions.

Crystalline II-26 was obtained using a vapor diffusion method. CompoundII-26 (15 mg) was dissolved in 100 μl of acetone in a 1.5 ml v-bottomHPLC vial. This vial was then placed inside a larger tightly-stopperedvessel containing 1 ml of pentane. Crystals suitable for X-raycrystallography experiments were observed along the sides and bottom ofthe inner vial after 48 hours of incubation at 4° C. Crystallographydata was collected on a Bruker SMART APEX CCD X-ray diffractometer(F(000)=2656, Mo_(Kα) radiation, λ=0.71073 Å, μ=0.264 mm⁻¹, T=100K) atthe UCSD Crystallography Facility and the refinement method used wasfull-matrix least-squares on F². Crystal data NPI-2065: C₁₅H₂₀ClNO₄,MW=313.77, tetragonal, space group P4(1)2(1)2, a=b=11.4901(3) Å,c=46.444(2) Å, α=β=γ=90°, vol=6131.6(3) Å³, Z=16, ρ_(calcd)=1.360 gcm⁻³, crystal size, 0.30×0.15×0.07 mm³, θ range, 1.75-26.00°, 35367reflections collected, 6025 independent reflections (R_(int)=0.0480),final R indices (I>2σ(I)): R₁=0.0369, wR₂=0.0794, GOF=1.060.

In order to separate II-28 from II-20, a reverse-phase isocratic methodwas employed. Sample (69.2 mg) containing both compounds was dissolvedin acetonitrile to a concentration of 10 mg/ml, and 500 μl was loaded ona reverse-phase HPLC column (ACE 5μ C18-HL, 15 cm×21 mm ID) perinjection. An isocratic solvent system of 27% acetonitrile/63% water atflow rate of 14.5 ml/min was used to separate compounds II-28 and II-20,which eluted after 14 and 16 minutes, respectively. Fractions containingcompounds of interest were immediately evaporated under reduced pressureat room temperature on a rotary evaporator. Samples were then loadedonto a small column of silica and eluted with 10 ml of 70% hexane/30%acetone to remove additional impurities.

Samples generated from the preparative normal-phase HPLC methoddescribed above that contained II-20 but were free of II-28 could alsobe triturated with 100% EtOAc to remove minor lipophilic impurities.

Starting Compound II-16: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. LowRes. Mass: m/z 314 (M+H), 336 (M+Na).

Compound of Formula II-20: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. LowRes. Mass: m/z 266 (M+H); HRMS (ESI), m/z 266.1396 (M+H), Δ_(calc)=1.2ppm. FIG. 16 depicts the 1H NMR spectrum of a compound having thestructure of Formula II-20.

Compound of Formula II-24C: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm.

Low Res. Mass: m/z 328 (M+H), 350 (M+Na); HRMS (ESI), m/z 328.1309(M+H), Δ_(calc)=−2.0 ppm, C₁₆H₂₃NO₄Cl. FIG. 19 depicts the 1H NMRspectrum of a compound having the structure of Formula II-24C.

Compound of Formula II-26: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm;HRMS (ESI), m/z 314.1158 (M+H), Δ_(calc)=−0.4 ppm, C₁₅H₂₁NO₄Cl; FIG. 23depicts the ¹H NMR spectrum of a compound having the structure ofFormula II-26 in DMSO-d₆).

Compound of Formula II-28: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm;HRMS (ESI), m/z 266.1388 (M+H), Δ_(calc)=−1.8 ppm, C₁₄H₂₀NO₄. FIG. 26depicts the ¹H NMR spectrum of a compound having the structure ofFormula II-28 in DMSO-d₆).

3B: Large Scale Purification of Compound II-16

A two-step process was developed for the purification of Compound II-16from crude extracts obtained from the culture broth of NPS21184 Thefirst step of the process involves normal phase flash chromatography ofthe crude extract to produce material highly enriched in Compound II-16(˜95% purity). The enriched material is further purified by multiplecrystallizations until no single impurity is present at ≧1.0% in thefinal colorless crystalline material of Compound II-16

Biotage Flash Chromatography (Purification Step 1)

The Biotage Flash 75Li system, including a Flash 75L KP-Sil cartridge,without SIM, was used for flash chromatography. Based on methoddevelopment experiments, the loading capacity of the system wasestablished to be 10 g of crude extract per 800 g of silica. Betterresolution of Compound II-16 and Compound II-17 may be obtained byloading 8 g of crude extract on 800 g of silica; however, the recoverywas comparable using either load.

Crude extract (10.0 g, dried by high vacuum) containing Compound II-16(5.30 g; estimated from standard plot under HPLC) in a 250 ml Erlenmeyerflask was dissolved to a concentration of 107 mg/ml in acetone (93.5 ml)by sonicating the sample at 40° C. for about 6 minutes. The sample wasfiltered by gravity using fluted filter paper into another 250 mlErlenmeyer flask, and the filter paper was washed with 5 ml of acetone.A 150 μl aliquot of this solution was transferred into a tared 1 dramvial for purity analysis and mass estimation of Compound II-16. Thefiltered crude extract was directly injected onto a new, dry Flash 75LKP-Sil cartridge using an open 60 ml syringe as a funnel and bytransferring the sample from flask to syringe using a glass volumetricpipette. Due to the low viscosity of the sample, gravity was sufficientto load the sample onto the column; no additional pressure was required.Once the entire sample was absorbed onto the column, the flask waswashed with 5 ml of acetone, and this solution was also loaded. Fiveminutes after loading the sample, the system was pressurized by houseair, and then the following solvent step gradient was run through thecolumn at approximately 15 psi and a flow rate between 235 ml/min and250 ml/min:

1. 3 CV* 10% EtOAc in n-Heptane

2. 15 CV* 25% EtOAc in n-Heptane

3. 5 CV* 30% EtOAc in n-Heptane

*For 75L cartridge, 1 CV=1070 ml

The eluent for the first step was collected as one fraction (˜2500 ml),after which 500 ml fractions (0.47 column volume) were collected. Inorder to determine which fractions contained Compound II-16, fractionswere analyzed by TLC. Each fraction was spotted onto a Si TLC plate,developed in 40% EtOAc/60% hexanes, and visualized usingphosphomolybidic acid spray, then heat. A 1 ml aliquot was subsequentlytaken from each fraction containing Compound II-16, dried under a streamof nitrogen, redissolved in 0.5 ml of ACN and analyzed by LC-MS. Whilesamples were being run on LC-MS, fractions were covered with aluminumfoil and allowed to stand at room temperature.

The resulting chromatograms (UV @ 210 nm) were analyzed by onlyintegrating Compound II-16, Compound II-26, and Compound II-17 peaks.Those fractions containing Compound II-16, as well as <10% CompoundII-26 and <5% Compound II-17, were pooled and concentrated down to 4-6%(˜600 mL) of the total pooled volumes using a Buchi R-220 Rotavapor withwater bath temperature between 28° C. and 30° C. The yellow liquid inthe flask was slowly siphoned out leaving behind the white solid. Thewhite solid was dissolved in acetone and transferred to a 2 L roundbottom flask and concentrated by a rotary evaporator at 30° C. andweighed. The dry aliquot was analyzed on LC-MS. The purity of the samplewas greater than 95% (95.7%) and the major impurities were CompoundII-26 (3.2%) and Compound II-17 (0.38%). The yield of Compound II-16 was86% after this step, as calculated from the mass of Compound II-16recovered from flash chromatography versus the mass of Compound II-16estimated to be present in the crude extract. Samples containingCompound II-16 were stored at −20° C. without desiccation until furtherpurification by crystallization.

When multiple Biotage runs are required to process quantities of crudeextract that exceed the loading capacity of a single column, the abovepurification process should be completed for each individual Biotagerun. Resulting material from each run may then be combined andcrystallized using the steps described below, in order to yield onefinal lot of Compound II-16.

Crystallization-General Procedure (Purification Step-2)

The Compound II-16 samples obtained from Biotage flash chromatographytypically contain about 3% of NPI-2065. The main aim of thecrystallization step is to increase the purity of NPI-0052 to >97% andto reduce NPI-2065 to <1%.

Crystallizations were performed on Compound II-16 samples obtained fromBiotage Runs by dissolving the solid (4.56 g) in 1:1 acetone:n-heptane(910 ml) to a final concentration of 5 g/L. The solvent was slowlyevaporated under reduced pressure (about 275 mbar) using a rotaryevaporator with vacuum controller (Buchi R-200 Rotavapor) and a waterbath temperature of about 30° C. until the solvent was reduced to about43% (±6%) of its original volume. During this evaporation process,crystals were formed around the flask walls as well as in the solution.The solution (supernatant) was removed by siphoning under house vacuuminto a 500 ml Erlenmeyer flask, concentrated by rotary evaporation andtransferred to a 20 ml scintillation vial by dissolving in acetone. Theacetone was removed under a stream of N₂, and the solid was furtherdried by high vacuum before it was weighed. The mass of the materialobtained from the supernatant was used to approximate the amount ofcrystalline material remaining. The crystals in the 2 L flask weredissolved in acetone (455 ml, the same volume of acetone used forinitial crystallization). In order to determine the purity, an aliquot(100 μl) was removed, concentrated under a stream of N₂, redissolved inACN to a final concentration of 1 mg/ml and analyzed by LC-MS.Analytical results showed a 31% reduction of Compound II-26. n-Heptane(455 ml; the same volume used for initial crystallization to make a 1:1acetone: n-heptane solution) was added to the 2 L flask and thecrystallization process was repeated. This process was reiterated untilCompound II-26 was reduced to <1%; 3-4 crystallizations were needed toreach this target.

The final crystallization was performed in EtOAC/n-heptane solution. Thecrystalline material obtained above was redissolved in 1:1EtOAc:n-heptane (same concentration as the first acetone:n-heptanecrystallization, i.e. 5 g/L). The solvent was slowly evaporated underreduced pressure (about 130 mbar) using a rotary evaporator with a waterbath temperature of 30° C. until the solvent was reduced to about 30% ofits original volume. During this evaporation process, most of thecrystals stayed suspended in solution and did not adhere strongly to thesides of the flask. The solution (supernatant) was removed by siphoningunder house vacuum into a 500 ml Erlenmeyer flask, and then concentratedby rotary evaporation in a 250 ml round bottom flask, dried by highvacuum pump and weighed. The crystals in the 2 L flask were also driedby high vacuum for about 2 hrs, after which the white, crystallinematerial was removed from the flask. A few crystals were randomlyselected from various places in the flask for purity analysis. TheCompound II-26 was reduced from 0.98% to 0.66% (average of two datapoints) in this step.

The yield was about 87% (±2) from crude extract to Biotagechromatography and about 56% (±6) from Biotage to crystallization) basedon two Biotage runs. The overall yield for these two crude extracts tofinal product was 43% and 54%. In addition compound II-16 wasconsistently obtained in >98% purity, with no single impurity ≧1%.Specifically, impurity Compound II-26 was reduced to 0.5% after fourrounds of crystallization. Finally, the material obtained from the lastcrystallization step, performed in EtOAc:n-heptane, resulted in crystalsthat were easy to manage in the solid state and these crystals can beeasily captured by filtration from the mother liquor.

3C: Purification of I-7

Supernatant material obtained from the crystallization step of the largescale purification of Compound II-16 described above was dissolved inacetone (80 mg/ml). Aliquots (500 ul) of this solution were injectedonto a normal-phase HPLC column using the conditions describedpreviously for normal phase purification of Compounds II-16, II-24C,II-26 and II-28. Compound of Formula I-7 eluted at 7.5 minutes as a purecompound.

Compound of Formula I-7 (FIG. 30): UV (Acetonitrile/H₂O) λ_(max) 225(sh)nm. Low Res. Mass: m/z 298 (M+H), 320 (M+Na).

Example 4 Fermentation of Starting Compounds II-17 and II-18 andCompound of Formula II-27

Strain CNB476 was grown in a 500-ml flask containing 100 ml of the firstvegetative medium consisting of the following per liter of deionizedwater: glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone, 5 g; andsynthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The firstseed culture was incubated at 28 degree C. for 3 days on a rotary shakeroperating at 250 rpm. Five ml of the first seed culture was inoculatedinto a 500-ml flask containing 100 ml of the second vegetative mediumconsisting of the following per liter of deionized water: starch, 10 g;yeast extract, 4 g; peptone, 2 g; ferric sulfate, 40 mg; potassiumbromide, 100 mg; calcium carbonate, 1 g; and sodium bromide, 30 g. Thesecond seed cultures were incubated at 28 degree C. for 7 days on arotary shaker operating at 250 rpm. Approximately 2 to 3 gram of sterileAmberlite XAD-7 resin were added to the second seed culture. The secondseed culture was further incubated at 28 degree C. for 2 days on arotary shaker operating at 250 rpm. Five ml of the second seed culturewas inoculated into a 500-ml flask containing 100 ml of the secondvegetative medium. The third seed culture was incubated at 28 degree C.for 1 day on a rotary shaker operating at 250 rpm. Approximately 2 to 3gram of sterile Amberlite XAD-7 resin were added to the third seedculture. The third seed culture was further incubated at 28 degree C.for 2 days on a rotary shaker operating at 250 rpm. Five ml of the thirdculture was inoculated into a 500-ml flask containing 100 ml of thesecond vegetative medium. The fourth seed culture was incubated at 28degree C. for 1 day on a rotary shaker operating at 250 rpm.Approximately 2 to 3 gram of sterile Amberlite XAD-7 resin were added tothe fourth seed culture. The fourth seed culture was further incubatedat 28 degree C. for 1 day on a rotary shaker operating at 250 rpm. Fiveml each of the fourth seed culture was inoculated into ten 500-ml flaskscontaining 100 ml of the second vegetative medium. The fifth seedcultures were incubated at 28 degree C. for 1 day on a rotary shakeroperating at 250 rpm. Approximately 2 to 3 grams of sterile AmberliteXAD-7 resin were added to the fifth seed cultures. The fifth seedcultures were further incubated at 28 degree C. for 3 days on a rotaryshaker operating at 250 rpm. Four ml each of the fifth seed culture wasinoculated into one hundred and fifty 500-ml flasks containing 100 ml ofthe production medium having the same composition as the secondvegetative medium. Approximately 2 to 3 grams of sterile Amberlite XAD-7resin were also added to the production culture. The production cultureswere incubated at 28 degree C. for 6 days on a rotary shaker operatingat 250 rpm. The culture broth was filtered through cheese cloth torecover the Amberlite XAD-7 resin. The resin was extracted with 2 times3 liters ethyl acetate followed by 1 time 1 liter ethyl acetate. Thecombined extracts were dried in vacuo. The dried extract, containing0.42 g of Starting Compound II-17 and 0.16 gram Compound of FormulaII-18, was then processed for the recovery of the compounds.

Example 5 Purification of Starting Compound II-17, II-18 and CompoundII-27

The pure Compounds II-17 and II-18 were obtained by reversed-phase HPLCas described below:

Column ACE 5 C18-HL Dimensions 15 cm × 21 mm ID Flow rate 14.5 ml/minDetection 214 nm Solvent Gradient of 35% Acetonitrile/65% H₂O to 90%Acetonitrile/10% H₂O over 15 min

Crude extract (100 mg) was dissolved in 15 ml of acetonitrile. Aliquots(900 ul) of this solution were injected onto a reversed-phase HPLCcolumn using the conditions described above. Compounds II-17 and II-18eluted at 7.5 and 9 minutes, respectively. Fractions containing the purecompounds were first concentrated using nitrogen to remove organicsolvent. The remaining solution was then frozen and lyophilized todryness.

An alternative purification method for Compound II-17 and II-18 wasdeveloped for larger scale purification and involved fractionation ofthe crude extract on a normal phase VLC column. Under these conditions,sufficient amounts of several minor metabolites were identified,including compound II-27. The crude extract (2.4 g) was dissolved inacetone (10 ml) and this solution adsorbed onto silica gel (10 cc) bydrying in vacuo. The adsorbed crude extract was loaded on a normal phasesilica VLC column (250 cc silica gel, column dimensions 2.5 cm diameterby 15 cm length) and washed with a step gradient of hexane/EtOAc,increasing in the percentage of hexane in steps of 5% (100 ml solventper step). The majority of Compound II-16 eluted in the 60% hexane/40%EtOAc wash while the majority of Compound II.17 eluted in the 50%hexane/50% ethyl acetate wash. Final separation of the compounds wasachieved using C18 HPLC chromatography (ACE 5μ C18-HL, 150 mm×21 mm ID)using an isocratic solvent system consisting of 35% ACN/65% H₂O. Underthese conditions, compound II-27 eluted at 11 minutes, compound II-17eluted at 12.00 minutes, traces of Compound II-16 eluted at 23.5minutes, and Compound II-18 eluted at 25.5 minutes. The resultingsamples were dried in vacuo using no heat to remove the aqueous solventmixture. The spectroscopic data for these samples of Compound II-16 andCompound II-18 were found to be identical with those of samples preparedfrom earlier purification methods. The sample of compound II-18 wasfound to contain 8% of the lactone hydrolysis product and was furtherpurified by washing through a normal phase silica plug (1 cm diameter by2 cm height) and eluting using a solvent mixture of 20% EtOAc/80%Hexanes (25 ml). The resulting sample was found to contain pure CompoundII-18.

The fractions containing compound II-27 described above were furtherpurified using normal phase semi preparative HPLC (Phenomenex Luna Si10μ, 100 Å; 250×10 mm id) using a solvent gradient increasing from 100%hexane to 100% EtOAc over 20 minutes with a flow rate of 4 ml/min.Compound II-27 eluted as a pure compound after 11.5 minutes (0.8 mg,0.03% isolated yield from dried extract weight).

Starting Compound II-17: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. HighRes. Mass (APCI): m/z 280.156 (M+H), Δ_(calc)=2.2 ppm, C₁₅H₂₂NO₄.

Compound II-18: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. High Res. Mass(APCI): m/z 358.065 (M+H), Δ_(calc)=−1.9 ppm, C₁₅H₂₁NO₄Br. 1H NMR inDMSO-d6 (see FIG. 14).

Compound II-27: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm; MS (HR-ESI),m/z 280.1556 (M+H) Δ_(calc)=2.7 ppm (C₁₅H₂₂NO₄); ¹H NMR (DMSO-d₆) seeFIG. 25.

Example 6 Preparation of Compound of Formula II-19 from StartingCompound II-16

A sample of Compound II-16 (250 mg) was added to an acetone solution ofsodium iodide (1.5 g in 10 ml) and the resulting mixture stirred for 6days. The solution was then filtered through a 0.45 micron syringefilter and injected directly on a normal phase silica HPLC column(Phenomenex Luna 10u Silica, 25 cm×21.2 mm) in 0.95 ml aliquots. TheHPLC conditions for the separation of compound formula II-19 fromunreacted Compound II-16 employed an isocratic HPLC method consisting of24% ethyl acetate and 76% hexane, in which the majority of compoundII-19 eluted 2.5 minutes before Compound II-16. Equivalent fractionsfrom each of 10 injections were pooled to yield 35 mg compound II-19.Compound II-19: UV (Acetonitrile/H₂O) 225 (sh), 255 (sh) nm; ESMS, m/z406.0 (M+H); HRMS (ESI), m/z 406.0513 [M+H]⁺, Δ_(calc)=−0.5 ppm,C₁₅H₂₁NO₄I; ¹H NMR in DMSO-d₆ (see FIG. 15).

Example 7 Synthesis of the Compounds of Formulae II-2, II-3, and II-4

Compounds of Formulae II-2, II-3 and II-4 can be synthesized fromStarting Compounds II-16, II-17, and II-18, respectively, by catalytichydrogenation.

Example 7A Catalytic Hydrogenation of Starting Compound II-16

Starting Compound II-16 (10 mg) was dissolved in acetone (5 mL) in ascintillation vial (20 mL) to which was added the 10% (w/w) Pd/C (1-2mg) and a magnetic stirrer bar. The reaction mixture was stirred in ahydrogen atmosphere at room temperature for about 15 hours. The reactionmixture was filtered through a 3 cc silica column and washed withacetone. The filtrate was filtered again through 0.2 μm Gelman Acrodiscto remove any traces of catalyst. The solvent was evaporated off fromfiltrate under reduced pressure to yield the compound of Formula II-2 asa pure white powder.

Example 7B Catalytic Hydrogenation of Starting Compound II-17

Starting Compound II-17 (5 mg) was dissolved in acetone (3 mL) in ascintillation vial (20 mL) to which was added the 10% (w/w) Pd/C (about1 mg) and a magnetic stirrer bar. The reaction mixture was stirred in ahydrogen atmosphere at room temperature for about 15 hours. The reactionmixture was filtered through a 0.2 μm Gelman Acrodisc to remove thecatalyst. The solvent was evaporated off from filtrate to yield thecompound of Formula II-3 as a white powder which was purified by normalphase HPLC using the following conditions:

-   -   Column: Phenomenex Luna 10u Silica    -   Dimensions: 25 cm×21.2 mm ID    -   Flow rate: 14.5 ml/min    -   Detection: ELSD    -   Solvent: 5% to 60% EtOAc/Hex for 19 min, 60 to 100% EtOAc in 1        min, then 4 min at 100% EtOAc

Compound of Formula II-3 eluted at 22.5 min as a pure compound.

Example 7C Catalytic Hydrogenation of Compound II-18

3.2 mg of Starting Compound II-18 was dissolved in acetone (3 mL) in ascintillation vial (20 mL) to which was added the 10% (w/w) Pd/C (about1 mg) and a magnetic stirrer bar. The reaction mixture was stirred inhydrogen atmosphere at room temperature for about 15 hours. The reactionmixture was filtered through a 0.2 μm Gelman Acrodisc to remove thecatalyst. The solvent was evaporated off from filtrate to yield thecompound of Formula II-4 as a white powder which was further purified bynormal phase HPLC using the following conditions:

-   -   Column: Phenomenex Luna 10u Silica    -   Dimensions: 25 cm×21.2 mm ID    -   Flow rate: 14.5 ml/min    -   Detection: ELSD    -   Solvent: 5% to 80% EtOAc/Hex for 19 min, 80 to 100% EtOAc in 1        min, then 4 min at 100% EtOAc

Compound of Formula II-4 eluted at 16.5 min as a pure compound.

Example 8 Structural Characterization

The structure of the compounds can be elucidated by various methods,including NMR, MS, and UV. FIGS. 1-6 provide spectral data from thesemethods. UV for compound of Formula II-2 in acetonitrile/H₂O: λ_(max)225 (sh) nm. FIG. 1 depicts the NMR spectrum of the compound of FormulaII-2 in DMSO-d6. FIG. 2 depicts the low resolution mass spectrum of thecompound of Formula II-2: m/z 316 (M+H), 338 (M+Na). UV for compound ofFormula II-3 in acetonitrile/H₂O: λ_(max) 225 (sh) nm. FIG. 3 depictsthe NMR spectrum of the compound of Formula II-3 in DMSO-d6. FIG. 4depicts the low resolution mass spectrum of the compound of FormulaII-3: m/z 282 (M+H), 304 (M+Na). UV for compound of Formula inacetonitrile/H₂O: λ_(max) 225 (sh) nm. FIG. 5 depicts the NMR spectrumof the compound of Formula II-4 in DMSO-d6. FIG. 6 depicts the lowresolution mass of the compound of Formula II-4: m/z 360 (M+H), 382(M+Na).

In addition, high resolution mass spectrometry data were obtained forcompounds II-2, II-3, and II-4. Compound II-2: HRMS (ESI), m/z 316.1305[M+H]⁺, Δ_(calc)=−3.5 ppm, C₁₅H₂₃NO₄Cl. Compound II-3: HRMS (ESI), m/z282.1706 [M+H]⁺, Δ_(calc)=0.3 ppm, C₁₅H₂₄NO₄. Compound II-4: HRMS (ESI),m/z 360.0798 [M+H]⁺, Δ_(calc)=−3.4 ppm, C₁₅H₂₃NO₄Br

Example 9 Synthesis of the Compounds of Formulae II-5A and II-5B

A compound of Formula II-5A and Formula II-5B can be synthesized fromStarting Compound II-16 by epoxidation with mCPBA.

Starting Compound II-16 (101 mg, 0.32 mmole) was dissolved inmethylenechloride (30 mL) in a 100 ml of round bottom flask to which wasadded 79 mg (0.46 mmole) of meta-chloroperbenzoic acid (mCPBA) and amagnetic stir bar. The reaction mixture was stirred at room temperaturefor about 18 hours. The reaction mixture was poured onto a 20 cc silicaflash column and eluted with 120 ml of CH₂Cl₂, 75 ml of 1:1 ethylacetate/hexane and finally with 40 ml of 100% ethyl acetate. The 1:1ethyl acetate/hexane fractions yield a mixture of diastereomers of epoxyderivatives, Formula II-5A and II-5B, which were separated by normalphase HPLC using the following conditions:

Column Phenomenex Luna 10 u Silica Dimensions 25 cm × 21.2 mm ID Flowrate 14.5 ml/min Detection ELSD Solvent 25% to 80% EtOAc/Hex over 19min, 80 to 100% EtOAc in 1 min, then 5 min at 100% EtOAc

Compound Formula II-5A (major product) and II-5B (minor product) elutedat 21.5 and 19 min, respectively, as pure compounds. Compound II-5B wasfurther chromatographed on a 3 cc silica flash column to remove tracesof chlorobenzoic acid reagent.

Chemical Structures

Structural Characterization

Formula II-5A: UV (Acetonitrile/H₂O) λ_(max) 225 (sh) nm. Low Res. Mass:m/z 330 (M+H), 352 (M+Na). HRMS (ESI), m/z 330.1099 [M+H]⁺,Δ_(calc)=−2.9 ppm, C₁₅H₂₁NO₅Cl. FIGS. 7 and 8, respectively depict the1H NMR spectrum of Formula II-5A and the mass spectrum of Formula II-5A.

Formula II-5B: UV (Acetonitrile/H₂O) λ_(max) 225 (sh) nm. Low Res. Mass:m/z 330 (M+H), 352 (M+Na). HRMS (ESI), m/z 330.1105 [M+H]⁺,Δ_(calc)=−0.9 ppm, C₁₅H₂₁NO₅Cl. FIGS. 9 and 10, respectively depict the1H NMR spectrum of II-5B and the mass spectrum of II-5B.

Example 10 Synthesis of the Compounds of Formulae IV-1, IV-2, IV-3 andIV-4 Synthesis of Diol Derivatives (Formula IV-2)

Diols can be synthesized by Sharpless dihydroxylation using AD mix-α andβ: AD mix-α is a premix of four reagents, K₂OsO₂(OH)₄; K₂CO₃; K₃Fe(CN)₆;(DHQ)₂-PHAL [1,4-bis(9-O-dihydroquinine)phthalazine] and AD mix-β is apremix of K₂OsO₂(OH)₄; K₂CO₃; K₃Fe(CN)₆; (DHQD)₂-PHAL[1,4-bis(9-O-dihydroquinidine)phthalazine] which are commerciallyavailable from Aldrich. Diol can also be synthesized by acid or basehydrolysis of epoxy compounds (Formula II-5A and II-5B) which can bedifferent to that of products obtained in Sharpless dihydroxylation intheir stereochemistry at carbons bearing hydroxyl groups

Sharpless Dihydroxylation of Starting Compounds II-16, II-17 and II-18

The starting compounds are dissolved in t-butanol/water in a roundbottom flask to which is added AD mix-α or β and a magnetic stir bar.The reaction are monitored by silica TLC as well as mass spectrometer.The pure diols are obtained by usual workup and purification by flashchromatography or HPLC. The structures are confirmed by NMR spectroscopyand mass spectrometry. In this method both hydroxyl groups are on sameside.

Nucleophilic Ring Opening of Epoxy Compounds (II-5)

The epoxy ring is opened with various nucleophiles like NaCN, NaN₃,NaOAc, HBr, etc. to substitute various groups on cyclohexane ring withhydroxyl group one side.

EXAMPLES

The epoxy is opened with HCl to make Formula IV-3:

Compound of Formula II-5A (3.3 mg) was dissolved in acetonitrile (0.5ml) in a 1 dram vial to which was added 5% HCl (500 ul) and a magneticstir bar. The reaction mixture was stirred at room temperature for aboutan hour. The reaction was monitored by mass spectrometry. The reactionmixture was directly injected on normal phase HPLC to obtain compound ofFormula IV-3C as a pure compound without any work up. The HPLCconditions used for the purification were as follows: Phenomenex Luna10u Silica column (25 cm×21.2 mm ID) with a solvent gradient of 25% to80% EtOAc/Hex over 19 min, 80 to 100% EtOAc in 1 min, then 5 min at 100%EtOAc at a flow rate of 14.5 ml/min. An ELSD was used to monitor thepurification process. Compound of Formula IV-3C eluted at about 18 min(2.2 mg). Compound of Formula IV-3C: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm; ESMS, m/z 366 (M+H), 388 (M+Na); HRMS (ESI), m/z 366.0875[M+H]⁺, Δ_(calc)=0.0 ppm, C₁₅H₂₂NO₅Cl₂; ¹H NMR in DMSO-d₆ (FIG. 21) Thestereochemistry of the compound of Formula IV-3C was determined based oncoupling constants observed in the cyclohexane ring in 1:1 C₆D₆/DMSO-d₆(FIG. 22).

Reductive ring opening of epoxides (II-5): The compound of Formula istreated with metalhydrides like BH₃-THF complex to make compound ofFormula IV-4.

Example 11 Synthesis of the Compounds of Formulae II-13C and II-8C

Compound II-16 (30 mg) was dissolved in CH₂Cl₂ (6 ml) in a scintillationvial (20 ml) to which Dess-Martin Periodinane (122 mg) and a magneticstir bar were added. The reaction mixture was stirred at roomtemperature for about 2 hours. The progress of the reaction wasmonitored by TLC (Hex:EtOAc, 6:4) and analytical HPLC. From the reactionmixture, the solvent volume was reduced to one third, absorbed on silicagel, poured on top of a 20 cc silica flash column and eluted in 20 mlfractions using a gradient of Hexane/EtOAc from 10 to 100%. The fractioneluted with 30% EtOAc in Hexane contained a mixture of rotamers ofFormula II-13 C in a ratio of 1.5:8.5. The mixture was further purifiedby normal phase HPLC using the Phenomenex Luna 10u Silica column (25cm×21.2 mm ID) with a solvent gradient of 25% to 80% EtOAc/Hex over 19min, 80 to 100% EtOAc over 1 min, holding at 100% EtOAc for 5 min, at aflow rate of 14.5 ml/min. An ELSD was used to monitor the purificationprocess. Compound of Formula II-13C eluted at 13.0 and 13.2 mins as amixture of rotamers with in a ratio of 1.5:8.5 (7 mg). Formula II-13C:UV (Acetonitrile/H₂O) λ_(max) 226 (sh) & 300 (sh) nm; ESMS, m/z 312(M+H)⁺, 334 (M+Na)⁺; HRMS (ESI), m/z 312.1017 [M+H]⁺, Δ_(calc)=4.5 ppm,C₁₅H₁₉NO₄Cl; ¹H NMR in DMSO-d₆ (see FIG. 13).

The rotamer mixture of Formula II-13C (4 mg) was dissolved in acetone (1ml) in a scintillation vial (20 ml) to which a catalytic amount (0.5 mg)of 10% (w/w) Pd/C and a magnetic stir bar were added. The reactionmixture was stirred in a hydrogen atmosphere at room temperature forabout 15 hours. The reaction mixture was filtered through a 0.2 μmGelman Acrodisc to remove the catalyst. The solvent was evaporated fromthe filtrate to yield compound of Formula II-8C as a colorless gum whichwas further purified by normal phase HPLC using a Phenomenex Luna 10uSilica column (25 cm×21.2 mm ID) with a solvent gradient of 25% to 80%EtOAc/Hex over 19 min, 80 to 100% EtOAc over 1 min, holding at 100%EtOAc for 5 min, at a flow rate of 14.5 ml/min. An ELSD was used tomonitor the purification process. Compound of Formula II-8C (1 mg)eluted at 13.5 min as a pure compound. Formula II-8C: UV(Acetonitrile/H₂O) λ_(max) 225 (sh) nm; ESMS, m/z 314 (M+H)⁺, 336(M+Na)⁺; RMS (ESI), m/z 314.1149 [M+H]⁺, Δ_(calc)=3.3 ppm, C₁₅H₂₁NO₄Cl;¹H NMR in DMSO-d₆ (See FIG. 12).

Example 12 Synthesis of the Compound of Formulae II-25 from II-13C

The rotamer mixture of Formula II-13C (5 mg) was dissolved in dimethoxyethane (monoglyme; 1.5 ml) in a scintillation vial (20 ml) to whichwater (15 μl (1% of the final solution concentration)) and a magneticstir bar were added. The above solution was cooled to −78° C. on a dryice-acetone bath, and a sodium borohydride solution (3.7 mg of NaBH₄ in0.5 ml of monoglyme (created to allow for slow addition)) was addeddrop-wise. The reaction mixture was stirred at −78° C. for about 14minutes. The reaction mixture was acidified using 2 ml of 4% HClsolution in water and extracted with CH₂Cl₂. The organic layer wasevaporated to yield mixture of compound of formulae II-25 and II-16 in a9.5:0.5 ratio as a white solid, which was further purified by normalphase HPLC using a Phenomenex Luna 10u Silica column (25 cm×21.2 mm ID).The mobile phase was 24% EtOAc/76% Hexane, which was held isocratic for19 min, followed by a linear gradient of 24% to 100% EtOAc over 1 min,and held at 100% EtOAc for 3 min; the flow rate was 25 ml/min. An ELSDwas used to monitor the purification process. Compound of Formula II-25(1.5 mg) eluted at 11.64 min as a pure compound. Compound of FormulaII-25: UV (Acetonitrile/H₂O) λ_(max) 225 (sh) nm; ESMS, m/z 314 (M+H)⁺,336 (M+Na)⁺; HRMS (ESI), m/z 314.1154 [M+H]⁺, Δ_(calc)=−0.6 ppm,C₁₅H₂₁NO₄Cl; ¹H NMR in DMSO-d₆ (see FIG. 20).

Example 13 Synthesis of the Compounds of Formulae II-31, II-32 and II-49from II-13C; and Compounds of Formulae II-33, II-34, II-35 and II-36from II-31 and II-32

A rotamer mixture of the Compound of Formula II-13C (20 mg) wasdissolved in acetone (4 ml) in a scintillation vial (20 ml) to which acatalytic amount (3 mg) of 10% (w/w) Pd/C and a magnetic stir bar wereadded. The reaction mixture was stirred at room temperature for about 15hours. The reaction mixture was filtered through a 0.2 μm GelmanAcrodisc to remove the catalyst. The solvent was evaporated from thefiltrate to yield a mixture of diastereomers of hydroxy derivatives ofFormulae II-31 and II-32 (1:1), and a minor compound II-49, which wereseparated by reversed phase HPLC using Ace 5u C18 column (150 mm×22 mmID) with a solvent gradient of 90% to 30% H₂O/Acetonitrile over 15 min,70 to 100% Acetonitrile over 5 min, holding at 100% Acetonitrile for 4min, at a flow rate of 14.5 ml/min. A diode array detector was used tomonitor the purification process. Compound II-31 (2 mg), II-32 (2 mg)and II-49 (0.2 mg) eluted at 10.6, 10.8 and 11.54 min, respectively, aspure compounds. II-31: UV (Acetonitrile/H₂O) λ_(max) 250 (sh) nm; ESMSm/z 328.1 (M+H)⁺& 350.0 (M+Na)⁺. II-32: UV (Acetonitrile/H₂O) λ_(max)250 (sh) nm; ESMS, m/z 328.1 (M+H)⁺& 350.0 (M+Na)⁺. II-49: UV(Acetonitrile/H₂O) λ_(max) 250 (sh) and 320 nm; ESMS, m/z 326.0 (M+H)⁺,343.1 (M+H₂O)⁺& 348.0 (M+Na)⁺.

In an alternate method, compounds II-31, II-32 and II-49 were separatedby normal phase HPLC using Phenomenex Luna 10u Silica column (25 cm×21.2mm ID) with a solvent gradient of 10% to 100% Hexane/EtOAc over 24 min,holding at 100% EtOAc for 3 min, at a flow rate of 14.5 ml/min. ELSD wasused to monitor the purification process.

The ketone of the compounds of formula II-31 and II-32 can be reduced byusing sodium borohydride at 0 to −10° C. in monoglyme solvent for about14 minutes. The reaction mixture can be acidified using 4% HCl solutionin water and extracted with CH₂Cl₂. The organic layer can be evaporatedto yield the mixtures of compounds of formulae II-33, II-34, II-35 andII-36 which can be separated by chromatographic methods.

Example 14 Synthesis of the Compound of Formula II-21 from II-19

Acetone (7.5 ml) was vigorously mixed with 5 N NaOH (3 ml) and theresulting mixture evaporated to a minimum volume in vacuo. A sample of100 μl of this solution was mixed with compound of Formula II-19 (6.2mg) in acetone (1 ml) and the resulting biphasic mixture vortexed for 2minutes. The reaction solution was immediately subjected to preparativeC18 HPLC. Conditions for the purification involved a linear gradient if10% acetonitrile/90% water to 90% acetonitrile/10% water over 17 minutesusing an Ace 5μ C18 HPLC column of dimensions 22 mm id by 150 mm length.Compound of Formula II-21 eluted at 9.1 minutes under these conditionsto yield 0.55 mg compound. Compound of Formula II-21: UV(Acetonitrile/H₂O) 225 (sh), ESMS, m/z 296.1 (M+H); ¹H NMR in DMSO-d₆(see FIG. 17).

Example 15 Synthesis of the Compound of Formula II-22 from II-19

A sample of 60 mg sodium propionate was added to a solution of compoundof Formula II-19 (5.3 mg) in DMSO (1 ml) and the mixture sonicated for 5minutes, though the sodium propionate did not completely dissolve. After45 minutes, the solution was filtered through a 0.45μ syringe filter andpurified directly using HPLC. Conditions for the purification involved alinear gradient if 10% acetonitrile/90% water to 90% acetonitrile/10%water over 17 minutes using an Ace 5μ C18 HPLC column of dimensions 22mm id by 150 mm length. Under these conditions, compound of FormulaII-22 eluted at 12.3 minutes to yield 0.7 mg compound (15% isolatedyield). UV (Acetonitrile/H₂O) 225 (sh), ESMS, m/z 352.2 (M+H); HRMS(ESI), m/z 352.1762 [M+H]⁺, Δ_(calc)=0.6 ppm, C₁₈H₂₆NO₆; ¹H NMR inDMSO-d₆ (see FIG. 18).

Example 16 Synthesis of the Compound of Formula II-29 from II-19

A sample of NaN₃ (80 mg) was dissolved in DMSO (1 ml) and transferred toa vial containing Compound II-19 (6.2 mg) which was contaminated withapproximately 10% Compound II-16 contaminant at 12.5 minutes (4.2 mg,85% yield). A 2.4 mg portion of compound II-29 was further purifiedusing additional C18 HPLC chromatography (ACE 5μ C18-HL, 150 mm×21 mmID) using an isocratic solvent gradient consisting of 35%acetonitrile/65% H₂O. Under these conditions compound II-29 eluted after20 minutes, while Compound II-16 eluted after 21.5 minutes. Theresulting sample consisted of 1.1 mg Compound II-29 was used forcharacterization in biological assays.

Compound II-29: UV (Acetonitrile/H₂O) 225 (sh), ESMS, m/z 321.1 (M+H);¹H NMR in DMSO-d₆ (see FIG. 27).

Example 17 Synthesis of the Compounds of Formulae II-37 and II-38 fromII-19

The compounds of Formulae II-37 and II-38 can be prepared from thecompound of Formula II-19 by cyano-de-halogenation orthiocyanato-de-halogenation, respectively. Compound II-19 can be treatedwith NaCN or KCN to obtain compound II-37. Alternatively, Compound II-19can be treated with NaSCN or KSCN to obtain compound II-38.

Synthesis of the Compound of Formula II-38 from II-19

The compound of formula II-19 (10.6 mg, 0.02616 mmol) was dissolved in1.5 ml of acetone in a scintillation vial (20 ml) to which sodiumthiocyanate (10.0 mg, 0.1234 mmol), triethylamine (5 μl, 0.03597 mmol)and a magnetic stir bar were added. The reaction mixture was stirred atroom temperature for 72 hours. The reaction mixture was concentrated invacuo to yield the compound II-38, which was purified by normal phaseHPLC using a Phenomenex Luna 10μ Silica column (25 cm×21.2 mm ID) with asolvent gradient of 0 to 95% H₂O/Acetonitrile over 21 min, at a flowrate of 14.5 ml/min. Diode array detector was used to monitor thepurification process. Compound II-38 (3.0 mg, 34% yield) eluted at 18.0min as a pure compound. II-38: UV (Acetonitrile/H₂O) λ_(max) 203 (sh)nm; ESMS m/z 337.1 (M+H)⁺& 359.1 (M+Na)⁻.

Example 18 Synthesis of the Compound of Formula II-39 from II-19

Thiols and thioethers of the Formula II-39 can be formed bydehalogenation of the compound of Formula II-19. Thiols (R═H) can beformed by treatment of Compound II-19 with NaSH, for example, whilethioethers (R=alkyl) can be formed by treatment of Compound II-19 withsalts of thiols, or alternatively, by treatment with thiols themselvesby running the reaction in benzene in the presence of DBU.

Example 19 Synthesis of the Compound of Formula II-40 from II-39

Sulfoxides (n=1) and sulfones (n=2) of the Formula II-40 can be formedby oxidation of thioethers of the Formula II-39, for example, withhydrogen peroxide or other oxidizing agents.

Example 20 Synthesis of the Compound of Formula II-41 from II-21

The compound of the Formula II-41 can be prepared by treatment of thecompound of Formula II-21 (or a protected derivative of II-21, where theC-5 alcohol or lactam NH are protected, for example) with methylsulfonyl chloride (mesyl chloride) in pyridine, for example, or bytreatment with mesyl chloride in the presence of triethylaminde. Othersulfonate esters can be similarly prepared.

Example 21 Synthesis of the Compound of Formula II-46 from II-19 orII-41

The alkene of the Formula II-46 can be prepared by dehydroiodination ofthe compound of Formula II-19, or by hydro-mesyloxy elimination of thecompound of Formula II-41, for example, by treatment with base.

Example 22 Synthesis of the Compound of Formula II-42A

Synthesis of boronic acids or esters, for example, the compound of theFormula II-42A, can be achieved as outlined in the retrosynthetic schemebelow. Hydroboration of the alkene of Formula II-46 gives thecorresponding alkyl borane, which can be converted to the correspondingboronic acid or ester, for example, the compound of the Formula II-42A.

Example 23 Synthesis of the Compound of Formula II-43A

The compound of the Formula II-43A can be prepared by treatment of thecompound of Formula II-19 with triphenyl phosphine to make a phosphorusylide, which can be treated with various aldehydes, for example,glyoxylic acid methyl ester, to make Formula II-43A.

Example 24 Synthesis of the Compound of Formula II-30 from II-19

A portion of CuI (100 mg) was placed in a 25 ml pear bottom flask andflushed with Ar gas for 30 minutes and Ar gas flow was maintainedthrough the flask throughout the course of the reaction. The vessel wascooled to −78° C. prior to addition of dry THF (5 ml) followed by theimmediate dropwise addition of a solution of methyllithium in dry THF(5.0 ml, 1.6 M) with vigorous stirring. A solution of Compound II-19 indry THF (12 mg Compound II-19, 1 ml THF) was added slowly to the cleardialkylcuprate solution and the resulting mixture stirred at −78° C. for1 hr. The reaction was quenched by washing the THF solution through aplug of silica gel (1 cm diameter by 2 cm length) along with furtherwashing using a solution of 50% EtOAc/50% hexanes (50 ml). The combinedsilica plug washes were dried in vacuo and subjected to further C18 HPLCpurification in 2 injections (ACE 5μ C18-HL, 150 mm×21 mm ID) using anisocratic solvent gradient consisting of 35% ACN/65% H₂O. Compound II-30eluted under these conditions at 23.5 minutes and yielded 2.4 mgmaterial (27% isolated yield) at 90.8% purity as measured by analyticalHPLC. An alternative normal phase purification method can be utilizedusing a Phenomenex Luna 10μ Silica column (25 cm×21.2 mm ID) with asolvent gradient consisting of 100% hexanes/ethyl acetate to 0% hexanesover 20 minutes. Compound II-30 eluted under these conditions at 16.5minutes and yielded 3.0 mg material (41% isolated yield) at 97.1% puritymeasured by analytical HPLC.

Compound II-30: UV (Acetonitrile/H₂O) 225 (sh), ESMS, m/z 294.1 (M+H);HRMS (ESI), m/z 294.1696 [M+H]⁺, Δ_(calc)=−3.2 ppm, C₁₆H₂₄NO₄; ¹H NMR inDMSO-d₆ (see FIG. 28).

Example 25 Synthesis of the Compound of Formulae II-44 and VI-1A fromII-16

The compound of Formula II-16 (30 mg, 0.096 mmol) was dissolved inCH₂Cl₂ (9 ml) in a scintillation vial (20 ml) to which triethylamine (40μl, 0.29 mmol), methyl-3-mercapto propionate (thiol, 250 μl) and amagnetic stir bar were added. The reaction mixture was stirred at roomtemperature for about 4 hours. The solvent was evaporated from thereaction mixture to yield a mixture of compounds of Formulae II-44 andVI-1A (19:1), which were separated by reversed phase HPLC using Ace 5uC18 column (150 mm×22 mm ID) with a solvent gradient of 35% to 90%H₂O/Acetonitrile over 17 min, 90 to 100% Acetonitrile over 1 min,holding at 100% Acetonitrile for 1 min, at a flow rate of 14.5 ml/min.Diode array detector was used to monitor the purification process.Compounds II-44 (20 mg) and VI-1A (1 mg) eluted at 11.68 and 10.88 min,respectively, as pure compounds. Compound II-44: UV (Acetonitrile/H₂O)λ_(max) 240 (sh) nm; ESMS m/z 434.0 (M+H)⁺ & 456.0 (M+Na)⁺. CompoundVI-1A: UV (Acetonitrile/H₂O) λ_(max) 220 (sh) nm; ESMS, m/z 398.0 (M+H)⁺& 420.0 (M+Na)⁺.

Example 26 Oxidation of Secondary Hydroxyl Group in Starting Compoundsand Reaction with Hydroxy or Methoxy Amines

The secondary hydroxyl group in the Starting Compounds is oxidized usingeither of the following reagents: pyridinium dichromate (PDC),pyridinium chlorochromate (PCC), Dess-Martin periodinane or oxalylchloride (Swern oxidation) (Ref: Organic Syntheses, collective volumesI-VII). Preferably, Dess-Martin periodinane can be used as a reagent forthis reaction. (Ref: Fenteany G. et al. Science, 1995, 268, 726-73). Theresulting keto compound is treated with hydroxylamine or methoxy amineto generate oximes.

EXAMPLES

Example 27 Reductive Amination of Keto-Derivative of Starting Compounds

The keto derivatives are treated with sodium cyanoborohydride (NaBH₃CN)in the presence of various bases to yield amine derivatives of theStarting Compounds, which are subsequently hydrogenated with 10%Pd/C, H₂to reduce double bond in cyclohexene ring.

EXAMPLE

Example 28 Cyclohexene Ring Opening

The Starting Compounds can be protected, for example, at the alcoholand/or at the lactam nitrogen positions, and treated with OsO₄ and NaIO₄in THF-H₂O solution to yield dial derivatives which are reduced to thealcohol with NaBH₄. The protecting groups can be removed at theappropriate stage of the reaction sequence to produce II-7 or II-6.

EXAMPLE

Example 29 Dehydration of Alcohol Followed by Aldehyde Formation atLactone-Lactam Ring Junction

A Starting Compound is dehydrated, for example, by treatment withmesylchloride in the presence of base, or, for example, by treatmentwith Burgess reagent or other dehydrating agents,. The resultingdehydrated compound is treated with OsO₄, followed by NaIO₄, oralternatively by ozonolysis, to yield an aldehyde group at thelactone-lactam ring junction.

Example 30 Oxidation of the Cyclohexene Ring to Produce Cyclohexadienesor a Phenyl Ring

A Starting Compound, such as the ketone of Formula II-13C, is treatedwith Pd/C to produce a cyclohexadiene derivative. The new double bondcan be at any position of the cyclohexene ring. The ketone can bereduced, for example, with sodium borohydride, to obtain thecorresponding secondary alcohol(s). Alternatively, the cyclohexadienederivative can be further treated, for example with DDQ, to aromatizethe ring to a phenyl group. Similarly, the ketone can be reduced, forexample, with sodium borohydride, to obtain the corresponding secondaryalcohol(s).

As an alternate method, the starting compound, such as the compound ofFormula II-49, can be treated, for example with TMSCl to producecyclohexadiene derivative. The cyclohexadiene derivative can be furthertreated, for example with DDQ, to aromatize the ring to a phenyl group.The OTMS on the phenyl group can be removed, for example, with acid orbase. Similarly, the ketone can be reduced, for example, with sodiumborohydride, to obtain the corresponding secondary alcohol(s).

Example 31 Various Reactions on Aldehyde Derivatives

Wittig reactions are performed on the aldehyde group of I-1 usingvarious phosphorus ylides [e.g., (triphenylphosphoranylidene)ethane] toyield an olefin. The double bond in the side chain is reduced bycatalytic hydrogenation.

EXAMPLE

Reductive amination is performed on the aldehyde group using variousbases (e.g., NH₃) and sodium cyanoborohydride to yield aminederivatives. Alternatively, the aldehyde is reduced with NaBH₄ to formalcohols in the side chain.

EXAMPLE

Organometallic addition reactions to the aldehyde carbonyl can beperformed to yield various substituted secondary alcohols. Examples:

Example 32 Synthesis of the Compound of Formula II-47 from II-17

The compound of Formula II-17 (25 mg, 0.0896 mmol) was dissolved inCH₂Cl₂ (9 ml) in a scintillation vial (20 ml) to which triethylamine (38μl, 0.27 mmol), methyl-3-mercapto propionate (thiol, 250 μl) and amagnetic stir bar were added. The reaction mixture was stirred at roomtemperature for about 4 hours. The solvent was evaporated from thereaction mixture to yield the compound of Formulae II-47, which wasfurther purified by normal phase HPLC using Phenomenex Luna 10u Silicacolumn (25 cm×21.2 mm ID) with a solvent gradient of 10% to 100%Hexane/EtOAc over 24 min, holding at 100% EtOAc for 3 min, at a flowrate of 14.5 ml/min. ELSD was used to monitor the purification process.Compound II-47 (15 mg) eluted at 10.98 min as pure compound. CompoundII-47: UV (Acetonitrile/H₂O) λ_(max) 240 (sh) nm; ESMS m/z 400.1 (M+H)⁺& 422.1 (M+Na)⁺.

Example 33 Synthesis of the Compound of Formulae II-48 and VI-1B fromII-16

The compound of Formula II-16 (15 mg, 0.048 mmol) was dissolved in 1:1ratio of ACN/DMSO (8 ml) in a scintillation vial (20 ml) to whichtriethylamine (40 μl, 0.29 mmol), Glutathione (44.2 mg, 0.144 mmol) anda magnetic stir bar were added. The reaction mixture was stirred at roomtemperature for about 3 hours. The solvent was evaporated from thereaction mixture to yield the compound of Formula II-48, which waspurified by reversed phase HPLC using Ace 5u C18 column (150 mm×22 mmID) with a solvent gradient of 10% to 70% H₂O/Acetonitrile over 15 min,70 to 100% Acetonitrile over 5 min, holding at 100% Acetonitrile for 4min, at a flow rate of 14.5 ml/min. Diode array detector was used tomonitor the purification process. Compound II-48 (10 mg) eluted as apure compound at 8.255 min. Compound II-48: UV (Acetonitrile/H₂O)λ_(max) 235 (sh) nm; ESMS m/z 621.0 (M+H)⁺. Compound II-48 was unstablein solution and converted to compound VI-1B which appeared as a mixtureof II-48 and VI-1B in the ratio of 7:3. Compound VI-1B: UV(Acetonitrile/H₂O) λ_(max) 235 (sh) nm; ESMS, m/z 585.2 (M+H)⁻.

Example 34 Synthesis of the Compound of Formula II-50 and VI-1C fromII-16

The compound of Formula II-16 (10 mg, 0.032 mmol) was dissolved inCH₂Cl₂ (9 ml) in scintillation vial (20 ml) to which triethylamine (26.5μl, 0.192 mmol), N-Acetyl-L-Cysteine methyl ester (17 mg, 0.096 mmol)and a magnetic stir bar were added. The reaction mixture was stirred atroom temperature for about 4 hours. The solvent was evaporated from thereaction mixture to yield the mixture of compounds of Formulae II-50 andVI-1C, which were further purified by normal phase HPLC using PhenomenexLuna 10u Silica column (25 cm×21.2 mm ID) with a solvent gradient of 10%to 100% Hexane/EtOAc over 24 min, holding at 100% EtOAc for 3 min, at aflow rate of 14.5 ml/min. ELSD was used to monitor the purificationprocess. Compounds II-50 (2 mg) and VI-1C (0.2 mg) were eluted at 10.39and 10.57 min, respectively as pure compounds. Compound II-50: UV(Acetonitrile/H₂O) λ_(max) 230 (sh) nm; ESMS m/z 491.1 (M+H)⁺ & 513.0(M+Na)⁺. Compound VI-1C: UV (Acetonitrile/H₂O) λ_(max) 215 (sh) nm; ESMSm/z 455.1 (M+H)⁺ & 577.0 (M+Na)⁺

Example 35 Growth Inhibition of Colon, Prostate, Breast Lung, Ovarian,Multiple Myeloma and Melanoma

Human colon adenocarcinoma (HT-29; HTB-38), prostate adenocarcinoma(PC-3; CRL-1435), breast adenocarcinoma (MDA-MB-231; HTB-26), non-smallcell lung carcinoma (NCI-H292; CRL-1848), ovarian adenocarcinoma(OVCAR-3; HTB-161), multiple myeloma (RPMI 8226; CCL-155), multiplemyeloma (U266; TIB-196) and mouse melanoma (B16-F10; CRL-6475) cellswere all purchased from ATCC and maintained in appropriate culturemedia. The cells were cultured in an incubator at 37° C. in 5% CO₂ and95% humidified air.

For cell growth inhibition assays, HT-29, PC-3, MDA-MB-231, NCI-H292,OVCAR-3 and B16-F10 cells were seeded at 5×10³, 5×10³, 1×10⁴, 4×10³,1×10⁴ and 1.25×10³ cells/well respectively in 90 μl complete media into96 well (Corning; 3904) black-walled, clear-bottom tissue culture platesand the plates were incubated overnight to allow cells to establish andenter log phase growth. RPMI 8226 and U266 cells were seeded at 2×10⁴and 2.5×10⁴ cells/well respectively in 90 μl complete media into 96 wellplates on the day of the assay. 20 mM stock solutions of the compoundswere prepared in 100% DMSO and stored at −80° C. The compounds wereserially diluted and added in triplicate to the test wells.Concentrations ranging from 6.32 μM to 632 pM were tested for II-2 andII-4. II-3 was tested at concentrations ranging from 20 μM to 6.32 nM.Formulae II-18 and II-19 were tested at concentrations ranging from 2 μMto 200 pM. Formula II-5A and Formula II-5B were tested at finalconcentrations ranging from 2 μM to 632 pM and 20 μM to 6.32 nMrespectively. The plates were returned to the incubator for 48 hours.The final concentration of DMSO was 0.25% in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(cm)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (XLfit 3.0,ID Business Solutions Ltd). Where the maximum inhibition of cell growthwas less than 50%, an EC₅₀ value was not determined.

The data in Table 1 summarize the growth inhibitory effects of FormulaeII-2, II-3, II-4, II-5A, II-5B, II-18 and II-19 against the humancolorectal carcinoma, HT-29, human prostate carcinoma, PC-3, humanbreast adenocarcinoma, MDA-MB-231, human non-small cell lung carcinoma,NCI-H292, human ovarian carcinoma, OVCAR-3, human multiple myelomas,RPMI 8226 and U266 and murine melanoma B16-F10 cell lines.

TABLE 1 EC₅₀ values of Formulae II-2, II-3, II-4, II-5A, II-5B, II-18and II-19 against various tumor cell lines EC₅₀ (nM)* Cell line II-2II-3 II-4 II-5A II-5B II-18 II-19 HT-29 129 ± 21  >20000 132 ± 36 67 ±17 1070 18 ± 7.8 11 ± 1.6 1210 PC-3 284 ± 110 >20000 204 ± 49 109 ± 15 1330 35 ± 5.6 29 ± 4.0 1790 MDA-MB-231 121 ± 23  >20000 114 ± 4   61 ±4.6 1040 16 ± 2.8 14 ± 3.2 957 NCI-H292 322 >20000 192 102 ± 19  992 2927 ± 3.8 395 >20000 213 1250 41 OVCAR-3 188 >20000 >6320 80 1320 >200024 251 >6320 64 >2000 20 RPMI 8226  49 >20000 57 36 326 6.3 5.9 45 >20000 51 29 328 6.3 7.1 U266  39 >20000 39 10 118 4.2 3.2 32 >20000 34  9 111 4.2 3.4 B16-F10 194 >20000 163 78 ± 11 1270 19 13 ±1.9 180 >20000 175 1140 36 *Where n ≧ 3, mean ± standard deviation ispresented

The EC₅₀ values indicate that the Formulae II-2, II-4, II-5A, II-5B,II-18 and II-19 were cytotoxic against the HT-29, PC-3, MDA-MB-231,NCI-H292, RPMI 8226, U266 and B16-F10 tumor cell lines. II-2, II-5A,II-5B and II-19 were also cytotoxic against the OVCAR-3 tumor cells.

Example 36 Growth Inhibition of Human Multiple Myeloma by Formulae I-7,II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-18, II-19, II-20,II-21, II-22, II-24C, II-25, II-26, II-28, II-29, II-30, II-31, II-32,II-38, IV-3C, II-44, VI-1A, II-47 and II-50; RPMI 8226 and U266 cells

The human multiple myeloma cell lines, RPMI 8226 (ATCC; CCL-155) andU266 (ATCC; TIB-196) were maintained in appropriate culture media. Thecells were cultured in an incubator at 37° C. in 5% CO₂ and 95%humidified air.

For cell growth inhibition assays, RPMI 8226 cells and U266 were seededat 2×10⁴ and 2.5×10⁴ cells/well respectively in 90 μl complete mediainto Corning 3904 black-walled, clear-bottom tissue culture plates. 20mM stock solutions of the compounds were prepared in 100% DMSO,aliquoted and stored at −80° C. The compounds were serially diluted andadded in triplicate to the test wells. The final concentration range ofFormula I-7, II-3, II-8C, II-5B, II-13C, II-20, II-21, II-22, II-24C,II-25, II-26, II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, VI-1A andII-47 were from 20 μM to 6.32 nM. The final concentration of FormulaII-18, II-19, II-44 and II-50 ranged from 632 nM to 200 pM. The finalconcentration range of Formula II-2, II-4 and II-5A were from 2 μM to632 pM. The final concentration of DMSO was 0.25% in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(em)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (generated byXLfit 3.0 or XLfit 4.0, ID Business Solutions Ltd).

The data in Table 2 summarize the growth inhibitory effects of FormulaeI-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-18, II-19, II-20,II-21, II-22, II-24, II-25, II-26, II-28, II-29, II-30, II-31, II-32,II-38, IV-3C, II-44, VI-1A, II-47 and II-50 against human multiplemyeloma cell lines, RPMI 8226 and U266.

TABLE 2 EC₅₀ values of Formulae I-7, II-2, II-3, II-4, II-5A, II-5B,II-8C, II-13C, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26,II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, II-44, VI-1A, II-47 andII-50 against RPMI 8226 and U266 cells RPMI 8226 U266 Compound EC₅₀ (nM)EC₅₀ (nM) Formula I-7   250 ND   240 Formula II-2   49 39   45 32Formula II-3 >20000 >20000 >20000 >20000 Formula II-4   57 39   51 34Formula II-5A   36 10   29 9 Formula II-5B   326 118   328 111 FormulaII-8C >20000 >20000 >20000 >20000 FormulaII-13C >20000 >20000 >20000 >20000 Formula II-18     6.3 4.2     6.3 4.2Formula II-19     5.9 3.2     7.1 3.4 Formula II-20 8510 ± 3260 310 442Formula II-21 >20000 6090 >20000 9670 Formula II-22  9720 2860  11200903 Formula II-24C  2320 1150  1640 825 FormulaII-25 >20000 >20000 >20000 >20000 Formula II-26  2230 1300  1610 829Formula II-28 >20000 >20000 >20000 >20000 Formula II-29  4280 624  69401420 Formula II-30  4900 889  4160 1240 Formula II-31 >20000 ND >20000Formula II-32 >20000 ND >20000 Formula II-38  2600 ND  1800 FormulaIV-3C  >20000* 7760 8290 Formula II-44   12 ND     8.8 Formula VI-1A 8400 ND  7800 Formula II-47 8000 ± 3400 ND Formula II-50   10 ND Wheren ≧ 3, mean EC₅₀ value ± standard deviation is presented; *n = 3,standard deviation not applicable; ND = not determined

The EC₅₀ values indicate that Formulae II-2, II-4, II-5A, II-5B, II-18,II-19, II-20, II-22, II-24C, II-26, II-29, and II-30 were cytotoxicagainst RPMI 8226 and U266 cells. Formulae I-7, II-38, II-44, VI-1A,II-47 and II-50 were cytotoxic against RPMI 8226 cells. Formula II-21and IV-3C were cytotoxic against U266 cells.

Example 37 Growth inhibition of MES-SA, MES-SA/Dx5, HL-60 and HL-60/MX2Tumor Cell Lines

Human uterine sarcoma (MES-SA; CRL-1976), its multidrug resistantderivative (MES-SA/Dx5; CRL-1977), human acute promyelocytic leukemiacells (HL-60; CCL-240) and its multidrug resistant derivative(HL-60/MX2; CRL-2257) were purchased from ATCC and maintained inappropriate culture media. The cells were cultured in an incubator at37° C. in 5% CO₂ and 95% humidified air.

For cell growth inhibition assays, MES-SA and MES-SA/Dx5 cells were bothseeded at 3×10³ cells/well in 90 μl complete media into 96 well(Corning; 3904) black-walled, clear-bottom tissue culture plates and theplates were incubated overnight to allow cells to establish and enterlog phase growth. HL-60 and HL-60/MX2 cells were both seeded at 5×10⁴cells/well in 90 μl complete media into 96 well plates on the day ofcompound addition. 20 mM stock solutions of the compounds were preparedin 100% DMSO and stored at −80° C. The compounds were serially dilutedand added in triplicate to the test wells. Concentrations ranging from6.32 μM to 2 nM were tested for II-2 and II-4. II-3 was tested atconcentrations ranging from 20 μM to 6.32 nM. Compound II-18 was testedat concentrations ranging from 2 μM to 632 pM. The plates were returnedto the incubator for 48 hours. The final concentration of DMSO was 0.25%in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(em)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (XLfit 3.0,ID Business Solutions Ltd). Where the maximum inhibition of cell growthwas less than 50%, an EC₅₀ value was not determined.

The multidrug resistant MES-SA/Dx5 tumor cell line was derived from thehuman uterine sarcoma MES-SA tumor cell line and expresses elevatedP-Glycoprotein (P-gp), an ATP dependent efflux pump. The data in Table 3summarize the growth inhibitory effects of Formulae II-2, II-3, II-4 andII-18 against MES-SA and its multidrug resistant derivative MES-SA/Dx5.Paclitaxel, a known substrate of the P-gp pump was included as acontrol.

TABLE 3 EC₅₀ values of Formulae II-2, II-3, II-4 and II-18 againstMES-SA and MES-SA/Dx5 tumor cell lines EC₅₀ (nM) Fold Compound MES-SAMES-SA/Dx5 change* II-2 193 220 1.0 155 138 II-3 >20000 >20000NA >20000 >20000 II-4 163 178 0.9 140 93 II-18 22 32 1.2 17 14Paclitaxel 5.6 2930 798    4.6 5210 *Fold change = the ratio of EC₅₀values (MES-SA/Dx5:MES-SA)

The EC₅₀ values indicate that II-2, II-4 and II-18 have cytotoxicactivity against both MES-SA and MES-SA/Dx5 tumor cell lines. Themultidrug resistant phenotype was confirmed by the observation thatPaclitaxel was ˜800 times less active against the resistant MES-SA/Dx5cells.

HL-60/MX2 is a multidrug resistant tumor cell line derived from thehuman promyelocytic leukemia cell line, HL-60 and expresses reducedtopoisomerase II activity. The data presented in Table 4 summarize thegrowth inhibitory effects of Formulae II-2, II-3, II-4 and II-18 againstHL-60 and its multidrug resistant derivative HL-60/MX2. Mitoxantrone,the topoisomerase II targeting agent was included as a control.

TABLE 4 EC₅₀ values of Formulae II-2, II-3, II-4 and II-18 against HL-60and HL-60/MX2 tumor cell lines EC₅₀ (nM) Fold Compound HL-60 HL-60/MX2change* II-2 237 142 0.7 176 133 II-3 >20000 >20000 NA >20000 >20000II-4 143 103 0.8 111 97 II-18 27 19 0.7 23 18 Mitoxantrone 42 134030.6   40 1170 *Fold change = the ratio of EC₅₀ values (HL-60/MX2:HL-60)

The EC₅₀ values indicate that II-2, II-4 and II-18 retained cytotoxicactivity against both HL-60 and HL-60/MX2 tumor cell lines. Themultidrug resistant phenotype was confirmed by the observation thatMitoxantrone was ˜30 times less active against the resistant HL-60/MX2cells.

The compounds disclosed herein have activity against drug resistantmultiple myeloma cell lines. For example, the compounds are activeagainst MM.1R and Doxorubicin-resistant Dox-40 cell lines. Furthermore,the compounds are active against cell lines obtained from human multiplemyeloma patients that have relapsed after multiple prior therapies withDexamethasone, Bortezomib, and thalidomide. Thus, the compounds areactive against drug resistant multiple myeloma including multiplemyeloma exhibiting resistance to doxorubicin, dexamethasone, bortezomib,and thalidomide.

Example 38 Inhibition of NF-κB-Mediated Luciferase Activity by FormulaeII-2, II-3, II-4, II-5A, II-5B, II8C, II-13C II-18, II-19, II-20, II-21,II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-44, VI-1Aand IV-3C; HEK293 NF-κB/Luciferase Reporter Cell Line

The HEK293 NF-κB/luciferase reporter cell line is a derivative of thehuman embryonic kidney cell line (ATCC; CRL-1573) and carries aluciferase reporter gene under the regulation of 5× NF-κB binding sites.The reporter cell line was routinely maintained in complete DMEM medium(DMEM plus 10% (v/v) Fetal bovine serum, 2 mM L-glutamine, 10 mM HEPESand Penicillin/Streptomycin at 100 IU/ml and 100 μg/ml, respectively)supplemented with 250 μg/ml G418. When performing the luciferase assay,the DMEM basal medium was replaced with phenol-red free DMEM basalmedium and the G418 was omitted. The cells were cultured in an incubatorat 37° C. in 5% CO2 and 95% humidified air.

For NF-κB-mediated luciferase assays, HEK293 NF-κB/luciferase cells wereseeded at 1.5×10⁴ cells/well in 90 μl phenol-red free DMEM completemedium into Corning 3917 white opaque-bottom tissue culture plates. ForFormula II-2, Formula II-4, Formula II-5A, and Formula II-18, a 400 μMstarting dilution was made in 100% DMSO and this dilution was used togenerate a 8-point half log dilution series. This dilution series wasfurther diluted 40× in appropriate culture medium and ten μl aliquotswere added to the test wells in triplicate resulting in final testconcentrations ranging from 1 μM to 320 pM. For Formulae II-3, II-5B,II-8C, II-13C, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28,II-29, II-30, VI-1A and IV-3C, a 8 mM starting dilution was made in 100%DMSO and the same procedure was followed as described above resulting infinal test concentrations ranging from 20 μM to 6.3 nM. For FormulaII-19 and II-44, a 127 μM starting dilution was made in 100% DMSO andthe final test concentrations ranged from 0.1 nM to 317 nM. For FormulaII-20, a 2.5 mM or 8 mM starting solution was made in 100% DMSO and thefinal test concentrations ranged from 6.3 μM to 2.0 nM or 20 μM to 6.3nM, respectively. The plates were returned to the incubator for 1 hour.After 1 hr pretreatment, 10 μl of a 50 ng/ml TNF-α solution, prepared inthe phenol-red free DMEM medium was added, and the plates were incubatedfor an additional 6 hr. The final concentration of DMSO was 0.25% in allsamples

At the end of the TNF-α stimulation, 100 μl of Steady Lite HTSluciferase reagent (Packard Bioscience) was added to each well and theplates were left undisturbed for 10 min at room temperature beforemeasuring the luciferase activity. The relative luciferase units (RLU)were measured by using a Fusion microplate fluorometer (PackardBioscience). The EC₅₀ values (the drug concentration at which 50% of themaximal relative luciferase activity is inhibited) were calculated inPrism (GraphPad Software) using a sigmoidal dose response, variableslope model.

NF-κB regulates the expression of a large number of genes important ininflammation, apoptosis, tumorigenesis, and autoimmune diseases. In itsinactive form, NF-κB complexes with IκB in the cytosol and uponstimulation, IκB is phosphorylated, ubiquitinated and subsequentlydegraded by the proteasome. The degradation of IκB leads to theactivation of NF-κB and its translocation to the nucleus. The effects ofFormulae II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-18, II-19,II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30,II-44, VI-1A and IV-3C on the activation of NF-κB were evaluated byassessing the NF-κB-mediated luciferase activity in HEK293 NF-κB/Luccells upon TNF-α stimulation.

Pretreatment of NF-κB/Luc 293 cells with Formulae II-2, II-4, II-5A,II-5B, II-18, II-19, II-20, II-21, II-22, II-24C, II-26, II-29, II-30and II-44 resulted in a dose-dependent decrease of luciferase activityupon TNF-α stimulation. The EC₅₀ values to inhibit NF-κB-mediatedluciferase activity are shown in Table 5 and demonstrate that Compoundsof Formulae II-2, II-4, II-5A, II-5B, II-18, II-19, II-20, II-21, II-22,II-24C, II-26, II-29, II-30 and II-44 inhibited NF-κB activity in thiscell-based assay.

TABLE 5 EC₅₀ values of Formulae II-2, II-3, II-4, II-5A, II-5B, II-8C,II-13C, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27,II-28, II-29, II-30, II-44, VI-1A and IV-3C from NF-κB-mediatedluciferase reporter gene assay Compound EC₅₀* Formula II-2 71 ± 20 nMFormula II-3 >20 μM >20 μM Formula II-4 67 nM 88 nM Formula II-5A 33 nM30 nM Formula II-5B 279 nM 261 nM Formula II-8C >20 μM >20 μM FormulaII-13C >20 μM >20 μM Formula II-18 9 nM 11 nM Formula II-19 7 nM 10 nMFormula II-20 849 ± 225 nM** Formula II-21 3.2 μM 2.7 μM Formula II-22 1μM 728 nM Formula II-24C 5.3 μM 3.2 μM Formula II-25 >20 μM >20 μMFormula II-26 4.3 μM 4.1 μM Formula II-27 >20 μM >20 μM FormulaII-28 >20 μM >20 μM Formula II-29 1.2 μM 1.4 μM Formula II-30 2.2 μM 2.2μM Formula II-44 17 ± 4 nM Formula VI-1A >20 μM >20 μM Formula IV-3C >20μM >20 μM *EC₅₀ values of two independent experiments are shown. Where n≧ 3 the mean EC₅₀ value ± standard deviation is represented. **The assayalso was performed with compound II-20, and resulted in an EC₅₀ value of154 nM, which value was not included in the calculation of the mean EC₅₀value.

Example 39 In vitro Inhibition of Proteasome Activity by Formulae I-7,II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-18, II-19, II-20,II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-31,II-32, II-38, IV-3C, II-44, VI-1A and II-47

All the compounds were prepared as 20 mM stock solution in DMSO andstored in small aliquots at −80° C. Purified rabbit muscle 20Sproteasome was obtained from CalBiochem or Boston Biochem. To enhancethe chymotrypsin-like activity of the proteasome, the assay buffer (20mM HEPES, pH7.3, 0.5 mM EDTA, and 0.05% Triton X100) was supplementedwith SDS resulting in a final SDS concentration of 0.035%. The substrateused was suc-LLVY-AMC, a fluorogenic peptide substrate specificallycleaved by the chymotrypsin-like activity of the proteasome. Assays wereperformed at a proteasome concentration of 1 μg/ml in a final volume of200 μl in 96-well Costar microtiter plates. Formulae II-2, II-4, II-18,II-19, II-21, II-22 and II-44 were tested as eight-point dose responsecurves with final concentrations ranging from 500 nM to 158 pM. FormulaeI-7, II-5A, II-5B, II-20, II-29, II-30 and II-38 were tested atconcentrations ranging from 1 μM to 0.32 nM. Formulae II-3 and VI-1Awere tested as an eight-dose response curve with final concentrationsranging from 10 μM to 3.2 nM. Formula II-47 was tested at concentrationsranging from 5 μM to 1.6 nM, while Formulae II-8C, II-13C, II-24C,II-25, II-26, II-27, II-28, II-31, II-32 and IV-3C were tested withfinal concentrations ranging from 20 μM to 6.3 nM. The samples wereincubated at 37° C. for five minutes in a temperature controlledFluoroskan Ascent 96-well microplate reader (Thermo Electron, Waltham,Mass.). During the preincubation step, the substrate was diluted 25-foldin SDS-containing assay buffer. After the preincubation period, thereactions were initiated by the addition of 10 μl of the dilutedsubstrate and the plates were returned to the plate reader. The finalconcentration of substrate in the reactions was 20 μM. Fluorescence ofthe cleaved peptide substrate was measured at λ_(ex)=390 nm andλ_(em)=460 nm. All data were collected every five minutes for more than1.5 hour and plotted as the mean of triplicate data points. The EC₅₀values (the drug concentration at which 50% of the maximal relativefluorescence is inhibited) were calculated by Prism (GraphPad Software)using a sigmoidal dose-response, variable slope model. To evaluate theactivity of the compounds against the caspase-like activity of the 20Sproteasomes, reactions were performed as described above except thatZ-LLE-AMC was used as the peptide substrate. Formulae II-3, II-4, II-5A,II-5B, II-8C, II-13C, II-18, II-20, II-21, II-22, II-24C, II-25, II-26,II-27, II-28, II-29, II-30, IV-3C, II-44 and VI-1A were tested atconcentrations ranging from 20 μM to 6.3 nM. Formula II-2 was tested atconcentrations ranging from 10 μM to 3.2 nM, while Formula II-19 wastested at concentrations ranging from 5 μM to 1.58 nM. For theevaluation of the compounds against the trypsin-like activity of theproteasome, the SDS was omitted from the assay buffer and Boc-LRR-AMCwas used as the peptide substrate. Formula II-20 was tested atconcentrations ranging from 1.6 nM to 5 μM. Formulae II-3, II-8C,II-13C, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30,IV-3C and VI-1A were tested at concentrations ranging from 20 μM to 6.3nM. For Formulae II-2 and II-5B the concentrations tested ranged from 10μM to 3.2 nM, while Formulae II-4, II-5A, II-18 and II-19 were tested atconcentrations ranging from 1 μM to 0.32 nM. Formula II-44 was tested atconcentrations ranging from 2 μM to 632 pM.

Results (EC₅₀ values) are shown in Table 6 and illustrate that among thetested compounds, Formulae II-5A, II-18, II-19, II-20, II-21, II-22,II-29, II-38 and II-44 are the most potent inhibitors of thechymotrypsin-like activity of the 20S proteasome with EC₅₀ valuesranging from 2 nM to 11 nM. Formulae I-7, II-2, II-4, II-5B, II-30 andII-47 inhibit the proteasomal chymotrypsin-like activity with EC₅₀values ranging from 13 nM to 88 nM, while the EC₅₀ values of FormulaeII-3, II-26 and VI-1A ranged from 207 nM to 964 nM. Formula II-13C,II-24C, II-27, II-28 and IV-3C inhibited the chymotrypsin-like activitywith EC₅₀ values ranging from 1.4 μM to 10.6 μM. EC₅₀ values forFormulae II-8C, II-25, II-31 and II-32 were greater than 20 μM. Underthe conditions tested, Formulae II-2, II-3, II-4, II-5A, II-5B, II-13C,II-18, II-19, II-20, II-21, II-22, II-24C, II-26, II-29, II-30, II-44and VI-1A were able to inhibit the trypsin-like activity of the 20Sproteasome. Formulae II-4, II-5A, II-18, II-19 and II-29 inhibited thecaspase-like activity with EC₅₀ values ranging from 213 nM to 850 nM,while Formulae II-2, II-5B, II-20, II-21, II-22, II-30, II-44 and VI-1Ahad EC₅₀ values ranging from 956 nM to 8.7 μM.

TABLE 6 Effects of Formulae I-7, II-2, II-3, II-4, II-5A, II-5B, II-8C,II-13C, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27,II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, II-44, VI-1A and II-47on the various enzymatic activities of purified rabbit 20S proteasomesEC₅₀ Values* Analog Chymotrypsin-like Trypsin-like Caspase-like FormulaI-7 52 ± 2 nM ND ND Formula II-2 18 nM 230 nM 1.3 μM 19 nM 230 nM 1.7 μMFormula II-3 964 nM 5.5 μM >20 μM 890 nM 7.7 μM >20 μM Formula II-4 13nM 107 nM 850 nM 15 nM 110 nM 637 nM Formula II-5A 6 nM 87 nM 535 nM 7nM 90 nM 438 nM Formula II-5B 88 nM 762 nM 3.8 μM 85 nM 716 nM 2.9 μMFormula II-8C >20 μM >20 μM >20 μM >20 μM >20 μM >20 μM Formula II-13C7.6 μM 8.6 μM >20 μM 8.8 μM 12.8 μM >20 μM Formula II-18 2.3 nM 14 nM286 nM 2 nM 14 nM 213 nM Formula II-19 3 nM 13 nM 573 nM 3 nM 15 nM 739nM Formula II-20 7.7 ± 3.0 nM 318 nM 1.4 μM 321 nM 1.4 μM Formula II-217 nM 720 nM 2.6 μM 8 nM 879 nM 2.3 μM Formula II-22 7 nM 308 nM 1.3 μM 3nM 289 nM 1.4 μM Formula II-24C 2.2 μM 3.3 μM >20 μM 2.0 μM 3.1 μM >20μM Formula II-25 >20 μM >20 μM >20 μM >20 μM >20 μM >20 μM Formula II-26349 nM 2.0 μM >20 μM 319 nM 3.0 μM >20 μM Formula II-27 1.4 μM >20μM >20 μM Formula II-28 3.2 μM >20 μM >20 μM 3.3 μM >20 μM >20 μMFormula II-29 6 nM 175 nM 535 nM 8 nM 254 nM 520 nM Formula II-30 21 nM905 nM 956 nM 21 nM 1.2 μM 1.3 μM Formula II-31 >20 μM** ND ND FormulaII-32 >20 μM** ND ND Formula II-38 3.4 ± 0.2 nM ND ND Formula IV-3C 4.9μM >20 μM >20 μM 10.6 μM >20 μM >20 μM Formula II-44 11 nM 55 nM 1.4 μM8.7 nM 54 nM 1.4 μM Formula VI-1A 274 nM 3.1 μM 7.9 μM 207 nM 3.0 μM 8.7μM Formula II-47 50 ± 10 nM ND ND *EC₅₀ values of one or two independentexperiments are shown. Where n ≧ 3 the mean EC₅₀ value ± standarddeviation is presented, **n = 3, standard deviation not applicable. ND =not determined

Example 40 The Effects of Formula II-16, Formula II-17, Formula II-20and Omuralide on the Chymotrypsin-Like Activity of 20S Proteasomes inRPMI 8226 Cells

RPMI 8226 (ATCC, CCL-155), the human multiple myeloma cell line, wascultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100units/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and10% heat inactivated fetal bovine serum at 37° C., 5% CO₂ and 95%humidified air. To evaluate the inhibitory effects on thechymotrypsin-like activity of the 20S proteasome, test compoundsprepared in DMSO were appropriately diluted in culture medium and addedto 2.5×10⁵/ml RMPI 8226 cells. For Formula II-16, the final testconcentrations ranged from 1 nM to 100 nM. For Formula II-17, FormulaII-20 and Omuralide (Calbiochem, San Diego, Calif.), the final testconcentrations ranged from 1 nM to 10 μM. DMSO was used as the vehiclecontrol at a final concentration of 0.1%. Following 1 hr incubation ofRMPI 8226 cells with the compounds, the cells were pelleted bycentrifugation at 2,000 rpm for 10 sec at room temperature and washed 3×with ice-cold 1× Dulbecco's Phosphate-Buffered Saline (DPBS, Mediatech,Herndon, Va.). DPBS washed cells were lysed on ice for 15 min in lysisbuffer (20 mM HEPES, 0.5 mM EDTA, 0.05% Triton X-100, pH 7.3)supplemented with protease inhibitor cocktail (Roche Diagnostics,Indianapolis, Ind.). Cell debris was pelleted by centrifugation at14,000 rpm for 10 min, 4° C. and supernatants (=cell lysates) weretransferred to a new tube. Protein concentration was determined by theBCA protein assay kit (Pierce Biotechnology, Rockford, Ill.). Thechymotrypsin-like activity of the 20S proteasome was measured by usingthe Suc-LLVY-AMC fluorogenic peptide substrate (Boston Biochem,Cambridge, Mass.) in the proteasome assay buffer (20 mM HEPES, 0.5 mMEDTA, pH 8.0) containing a final concentration of 0.035% SDS. Thereactions were initiated by the addition of 10 μL of 0.4 mM Suc-LLVY-AMC(prepared by diluting a 10 mM solution of the peptide in DMSO 1:25 withassay buffer) to 190 μL of the cell lysates and incubated in the ThermoLab Systems Fluoroskan plate reader at 37° C. The released coumarin(AMC) was measured fluorometrically by using λ_(ex)=390 nm andλ_(em)=460 nm. The assay was performed in a microtiter plate (Corning3904) and followed kinetically with measurements every five minutes for2 hr. The total amount of protein used for each assay was 20 μg. Thefinal concentration of Suc-LLVY-AMC and DMSO was 20 μM and 0.2%,respectively. Results are presented as the percent inhibition of the 20Sproteasome chymotrypsin-like activity relative to the DMSO control.

Results in Table 7 show that exposure of RPMI 8226 cells to FormulaII-16, Formula II-17, Formula II-20 and Omuralide resulted in inhibitionof the chymotrypsin-like activity of the 20S proteasomes. Among them,Formula II-16 inhibits 85±7% of the chymotrypsin-like activity of the20S proteasome at 5 nM. At 100 nM, Formula II-16 is able to completelyinhibit the chymotrypsin-like activity of the 20S proteasome. At 100 nM,Formula II-17, Formula II-20 and Omuralide are only able to inhibit thechymotrypsin-like activity at 30±4%, 66±3% and 32±8%, respectively.

TABLE 7 Determination of the chymotrypsin-like activity of 20Sproteasomes derived from RMPI 8226 cells treated with Formula II-16,Formula II-17, Formula II-20 and Omuralide % inhibition of thechymotrypsin-like activity of 20S proteasomes in RPMI 8226 cell lysates(mean ± SD, n = 3) Compound 10,000 nM 1,000 nM 500 nM 100 nM 50 nM 10 nM5 nM 1 nM II-16 ND ND ND 98 ± 1 97 ± 0 94 ± 3 85 ± 7 30 ± 7  II-17 65 ±5 46 ± 4 39 ± 3 30 ± 4 26 ± 5  6 ± 6 10 ± 5 6 ± 6 II-20 87 ± 4 73 ± 2 71± 2 66 ± 3 64 ± 3 37 ± 3 31 ± 9  3 ± 10 Omuralide 93 ± 1 80 ± 8  68 ± 1132 ± 8  17 ± 11  4 ± 9  8 ± 9 5 ± 9 ND; not determined

Example 41 The effects of Formula II-16, Formula II-17, Formula II-20and Omuralide on the Chymotrypsin-Like Activity of 20S Proteasomes inPC-3 Cells

PC-3 (ATCC, CRL-1435), the human prostate cancer cell line, was culturedin F12K medium supplemented with 2 mM L-glutamine, 100 units/mlpenicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal bovineserum at 37° C., 5% CO₂ and 95% humidified air. To evaluate theinhibitory effects on the chymotrypsin-like activity of the 20Sproteasome, test compounds prepared in DMSO were appropriately dilutedin culture medium and added to 1.25×10⁵/ml PC-3 cells. For FormulaII-16, the final test concentrations ranged from 1 nM to 50 nM. ForFormula II-17, Formula II-20 and Omuralide (Calbiochem, San Diego,Calif.), the final test concentrations ranged from 1 nM to 10 μM. DMSOwas used as the vehicle control at a final concentration of 0.1%.Following 1 hr incubation of PC-3 cells with the compounds, the cellswere washed 3× with ice-cold 1× Dulbecco's Phosphate-Buffered Saline(DPBS, Mediatech, Herndon, Va.). DPBS washed cells were lysed on ice for15 min in lysis buffer (20 mM HEPES, 0.5 mM EDTA, 0.05% Triton X-100, pH7.3) supplemented with protease inhibitor cocktail (Roche Diagnostics,Indianapolis, Ind.). Cell debris was pelleted by centrifugation at14,000 rpm for 10 min, 4° C. and supernatants (=cell lysates) weretransferred to a new tube. Protein concentration was determined by theBCA protein assay kit (Pierce Biotechnology, Rockford, Ill.). Thechymotrypsin-like activity of the 20S proteasome was measured by usingthe Suc-LLVY-AMC fluorogenic peptide substrate (Boston Biochem,Cambridge, Mass.) in the proteasome assay buffer (20 mM HEPES, 0.5 mMEDTA, pH 8.0) containing a final concentration of 0.035% SDS. Thereactions were initiated by the addition of 10 μL of 0.4 mM Suc-LLVY-AMC(prepared by diluting a 10 mM solution of the peptide in DMSO 1:25 withassay buffer) to 190 μL of the cell lysates and incubated in the ThermoLab Systems Fluoroskan plate reader at 37° C. The released coumarin(AMC) was measured fluorometrically by using λ_(ex)=390 nm andλ_(em)=460 nm. The assay was performed in a microtiter plate (Corning3904) and followed kinetically with measurements every five minutes for2 hr. The total amount of protein used for each assay was 20 μg. Thefinal concentration of Suc-LLVY-AMC and DMSO was 20 μM and 0.2%,respectively. Results are presented as the percent inhibition of the 20Sproteasome chymotrypsin-like activity relative to the DMSO control.

Results in Table 8 show that exposure of PC-3 cells to Formula II-16,Formula II-17, Formula II-20 and Omuralide resulted in inhibition of thechymotrypsin-like activity of the 20S proteasomes similar to resultsobtained from RPMI 8226 cell-based experiments. Formula II-16 inhibits69% of the chymotrypsin-like activity of the 20S proteasome at 5 nM. At50 nM, Formula II-16 is able to completely inhibit the chymotrypsin-likeactivity of the 20S proteasome. At 100 nM, Formula II-17, Formula II-20and Omuralide inhibit the chymotrypsin-like activity at 26%, 57% and36%, respectively.

TABLE 8 Determination of the chymotrypsin-like activity of 20Sproteasomes derived from PC-3 cells treated with Formula II-16, FormulaII-17, Formula II-20 and Omuralide % inhibition of the chymotrypsin-likeactivity of 20S proteasomes in PC-3 cell lysates 10,000 1,000 CompoundnM nM 100 nM 50 nM 10 nM 5 nM 1 nM II-16 ND ND ND 98 ND 69 19 II-17 7949 26 ND 16 ND ND II-20 90 71 57 ND 38 ND ND Omuralide 90 80 36 ND 18 NDND ND: not determined

Example 42 Growth Inhibition of Human Multiple Myeloma, RPMI 8226, HumanColon Adenocarcinoma, HT-29 and Murine Melanoma, B16-F10 Cells in MediaContaining 1% or 10% Serum

The growth inhibitory activity of Formulae II-16, II-17 and FormulaII-18 against human multiple myeloma, RPMI 8226, human colonadenocarcinoma, HT-29 and mouse melanoma, B16-F10 cells in the presenceof 1% or 10% fetal bovine serum (FBS) was determined.

RPMI 8226 (CCL-155), HT-29 (HTB-38), and B16-F10 (CRL-6475) cells werepurchased from ATCC. RPMI 8226 cells were maintained in RPMI 1640 mediasupplemented with 10% (v/v) FBS, 2 mM L-glutamine, 1 mM sodium pyruvateand Penicillin/Streptomycin at 100 IU/ml and 100 μg/ml, respectively.HT-29 cells were maintained in McCoys 5A supplemented with 10% (v/v)FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% (v/v) non-essentialamino acids, 10 mM HEPES and Penicillin/Streptomycin at 100 IU/ml and100 μg/ml, respectively. B16-F10 cells were maintained in DMEMsupplemented with 10% (v/v) FBS, 2 mM L-glutamine, 10 mM HEPES andPenicillin/Streptomycin at 100 IU/ml and 100 μg/ml, respectively. Thecells were cultured in an incubator at 37° C. in 5% CO₂ and 95%humidified air.

For cell growth inhibition assays, HT-29 and B16-F10 cells were seeded5×10³, and 1.25×10³ cells/well respectively in 90 μl media containing10% (v/v) FBS or 1% (v/v) FBS into 96 well (Corning; 3904) black-walled,clear-bottom tissue culture plates. The plates were incubated overnightto allow cells to establish and enter log phase growth. RPMI 8226 cellswere seeded at 2×10⁴ cells/well in 90 μl RPMI media containing 10% (v/v)FBS or 1% (v/v) FBS into 96 well black-walled, clear-bottom tissueculture plates. 20 mM stock solutions of Formulae II-16, II-17 andFormula II-18 were prepared in 100% DMSO, aliquoted and stored at −80°C. Formulae II-16, II-17 and Formula II-18 were serially diluted inmedia containing 1% or 10% FBS and added in triplicate to the testwells. The final concentration of Formula II-16 ranged from 2 μM to 200pM. The final concentration range of Formula II-17 was from 20 μM to 6.3nM. The final concentration of Formula II-18 ranged from 2 μM to 630 pM.The plates were returned to the incubator for 48 hours. The finalconcentration of DMSO was 0.25% in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(em)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (generated byXLfit 3.0, ID Business Solutions Ltd).

The data in Table 9 summarize the growth inhibitory effects of FormulaeII-16, II-17 and Formula II-18 against the human multiple myeloma cellline, RPMI 8226 in media containing 1% or 10% FBS.

TABLE 9 EC₅₀ values of Formulae II-16, II-17 and Formula II-18 againstRPMI 8226 cells in media containing 1% or 10% FBS Compound 1% FBS, EC₅₀(nM) 10% FBS, EC₅₀ (nM) II-16 6.2 12 6.8 9.6 Mean 6.5 11 II-17 1100 30001300 2300 Mean 1200 2700 II-18 15 20 13 20 Mean 14 20

The EC₅₀ values indicate that Formulae II-16, II-17 and Formula II-18were cytotoxic against RPMI 8226 cells in media containing 1% or 10%FBS. There was a less than three fold decrease in the mean EC₅₀ ofFormulae II-16, II-17 and Formula II-18 when tested in media containing10% FBS relative to media containing 1% FBS.

The data in Table 10 summarize the growth inhibitory effects of FormulaII-16 against the human colon adenocarcinoma, HT-29 and the murinemelanoma, B16-F10 cell lines in media containing 1% or 10% FBS.

TABLE 10 Mean EC₅₀ values of Formula II-16 against HT-29 and B16-F10cells in media containing 1% or 10% FBS HT-29, EC₅₀ B16-F10, EC₅₀ (nM)mean ± SD (nM) mean ± SD Compound 1% FBS 10% FBS 1% FBS 10% FBS II-16 16± 5 23 ± 10 18 ± 9 13 ± 1

The mean EC₅₀ values indicate that Formula II-16 was cytotoxic againstHT-29 and B16-F10 cells in media containing 1% or 10% FBS. There was aless than two fold decrease in the mean EC₅₀ of Formula II-16 whentested in media containing 10% FBS relative to media containing 1% FBS.Taken together, these data show that with respect to the in vitrocytotoxic activity against tumor cell lines, Formulae II-16, II-17 andFormula II-18 maintain similar biological activity in the presence of 1%or 10% FBS.

Example 43

Anthrax toxin is responsible for the symptoms associated with anthrax.In this disease, B. anthracis spores are inhaled and lodge in the lungswhere they are ingested by macrophages. Within the macrophage, sporesgerminate, replicate, resulting in killing of the cell. Before killingoccurs, however, infected macrophages migrate to the lymph nodes where,upon death, they release their contents, allowing the organism to enterthe bloodstream, further replicate, and secrete lethal toxins.

Two proteins called protective antigen (PA; 83 kDa) and lethal factor(LF; 90 kDa), play a key role in the pathogenesis of anthrax. Theseproteins are collectively known as lethal toxin (LeTx). When combined,PA and LF cause death when injected intravenously in animals. Lethaltoxin is also active in a few cell culture lines of macrophages causingcell death within a few hours. LeTx can induce both necrosis andapoptosis in mouse macrophage-like RAW264.7 cells upon in vitrotreatment.

In Vitro Cell-Based Assay for Inhibitors of Lethal Toxin-MediatedCytotoxicity

RAW264.7 cells (obtained from the American Type Culture Collection) wereadapted to and maintained in RPMI-1640 medium supplemented with 10%fetal bovine serum, 2 mM L-glutamine and 1% Penicillin/Streptomycin(complete medium) at 37° C. in a humidified 5% CO₂ incubator. For theassay, cells were plated overnight in complete medium at a concentrationof 50,000 cells/well in a 96-well plate. Media was removed the followingday and replaced with serum-free complete medium with or without varyingconcentrations of Formulae II-2, II-3, II-4, II-5A, II-5B, II-13C, II-18and IV-3C starting at 330 nM and diluting at ½ log intervals for an8-point dose-response. After a 45 minute preincubation, 1 μg/ml LF and 1μg/ml PA alone or in combination (LF:PA, also termed lethal toxin(LeTx)) were added to cells. Recombinant LF and PA were obtained fromList Biological Laboratories. Additional plates with no LeTx added wereincluded as a control. Cells were then incubated for six hours followedby addition of 0.02 mg/ml resazurin dye (Molecular Probes, Eugene,Oreg.) prepared in Mg++, Ca++ free PBS (Mediatech, Herndon, Va.). Plateswere then incubated an additional 1.5 hours prior to the assessment ofcell viability. Since resazurin is metabolized by living cells,cytotoxicity or cell viability can be assessed by measuring fluorescenceusing 530 excitation and 590 emission filters. Data are expressed as thepercent viability as compared to a DMSO alone control (high) and theLeTx alone control (low) using the following equation: Percentviability=100*((Measured OD-low control)/(high control-low control)).

Inhibition of Anthrax Lethal Toxin-Mediated Cytotoxicity in RAW 264.7Cells

Data in FIG. 11 summarize the effects of Formula II-2, Formula II-3 andFormula II-4 against LeTx-mediated cytotoxicity of the RAW 264.7 murinemacrophage-like cell line. Treatment of RAW 264.7 cells with FormulaII-2 and Formula II-4 resulted in an increase in the viability of LeTxtreated cells with EC₅₀ values of 14 nM (FIG. 11). The EC₅₀ values forFormula II-3 for LeTx protection was not be determined at theconcentrations tested (EC₅₀>330 nM, the maximum concentrationevaluated). Data in Table 11 show the effects of Formulae II-5A, II-5B,II-13C, II-18 and IV-3C against LeTx-mediated cytotoxicity of the RAW264.7 murine macrophage-like cell line. Treatment of RAW 264.7 cellswith Formula II-5A and II-18 showed an increase in the viability of LeTxtreated RAW 264.7 cells with EC₅₀ values of 3 nM and 4 nM respectively.Treatment with Formula II-5B resulted in an increase in the viability ofLeTx treated cells with an EC₅₀ value of 45 nM. The EC₅₀ values forFormulae II-13C and IV-3C for LeTx protection could not be determined atthe concentrations tested (EC₅₀>330 nM, the maximum concentrationevaluated).

TABLE 11 EC₅₀ values for inhibition of RAW 264.7 cell cytotoxicitymediated by anthrax lethal toxin Compound EC₅₀ (nM) Formula II-18 4Formula II-5A 3 Formula II-5B 45  Formula II-13C >330 nM FormulaIV-3C >330 nM

Example 44 Structure-Activity Relationship of the R₁ Side Chain

A structure-activity relationship for the R₁ side chain of the disclosedcompounds and specifically the compounds of Formulas (I), (II), (III),(IV), and (V) was inferred by analyzing the relative activity of variouscompounds having the formula:

These compounds were assayed for cytotoxicity of RPMI cells and forinhibition of NF-κB and the chymotrypsin-, trypsin-, and caspase-likeactivities of the 20S proteasome. The results of 6 representativecompounds are presented in Table 12.

TABLE 12 EC₅₀ values for compounds having varying R₁ groups ProteasomeCytotoxicity Proteasome Trypsin- Proteasome RPMI EC₅₀ NF-κBChymotrypsin- like EC₅₀ Caspase-like Compound R₁ (nM) EC₅₀ (nM) likeEC₅₀ (nM) (nM) EC₅₀ (nM) II-16 CH₂CH₂Cl 7 ± 0.4 11 ± 3  2.6 ± 0.2 21 ±2.6 401 ± 93 II-18 CH₂CH₂Br 6.3, 6.3 11, 9 2.3, 2 14, 14 286, 213 II-19CH₂CH₂I 6, 7 10, 7 3, 3 13, 15 573, 739 II-17 CH₂CH₃ 6150, 3460 960 ±210  26 ± 6.7 573, 602 1247, 1206 II-20 CH₃ 8510 ± 3260 849 ± 225 7.7 ±3.0 318, 321 1425, 1420 II-21 CH₂CH₂OH >20000, 3172, 2707 7, 8 720, 8792585, 2328 >20000

The results of the above assays can be interpreted to suggest thatcompounds having R₁ groups of chloroethyl, bromoethyl, or iodoethyl arepotent inhibitors of proteasome and exhibit very potent cytotoxicity. Incontrast, compounds having R₁ groups of methyl, ethyl, or hydroxyethylexhibited relatively lower cytotoxicity (3-log decrease in potency),lower NF-κB inhibition (3-log decrease in potency), and a lowercaspase-like (2 to 10 fold less potent) and trypsin-like (20 to 50 foldless potent) proteasome inhibition.

Without being bound to any particular theory, the Applicants note thatthe above results support the hypothesis that the increased activity ofcompounds containing Cl, Br, or I in the R₁ group can be due to thehalogen's property of being a good leaving group. This hypothesis issupported by the fact that lactone ring opening of compound II-16 isobserved to form a cyclic ether through nucleophilic substitution wherechlorine is displaced according to the following reaction:

It is hypothesized that in compounds having a good leaving group in theR₁ side chain, such as compounds II-16, II-18, and II-19, nucleophilicaddition of the proteasome to the β-lactone ring forms a cyclic ether ina manner similar to the above reaction. The cyclic ether, or formationthereof, is hypothesized to interact favorably with the proteasome.

Without being bound to any particular theory, the Applicants note thatthe above results also support the alternative hypothesis that a secondnucleophile on the proteasome displaces the leaving group, thus forminga 2-point covalent adduct between the compound and the enzyme. In eithercase, leaving group functionality on the R₁ side chain promotesincreased interaction between the compound and the enzyme and thuspromotes increased activity. Therefore, compounds having other leavinggroups on the R₁ side chain can be expected to exhibit high activity.

Without being bound to any particular theory, the Applicants note thatthe above results also support the hypothesis of a single-point leavinggroup. As one example, the presence of a halogen or other leaving groupin the R₁ side chain promotes the delivery of the compound to itstarget, such as an intracellular or other biological target, therebyenhancing its therapeutic effect. An example of a single-point leavinggroup is illustrated in the diagram shown below.

“Leaving groups” as used herein refers to any atom or moiety that iscapable of being displaced by another atom or moiety in a chemicalreaction. More specifically, in some embodiments, “leaving group” refersto the atom or moiety that is displaced in a nucleophilic substitutionreaction. In some embodiments, “leaving groups” are any atoms ormoieties that are conjugate bases of a strong acid. Non-limitingcharacteristics and examples of leaving groups can be found, for examplein Organic Chemistry, 2d ed., Francis Carey (1992), pages 328-331;Introduction to Organic Chemistry, 2d ed., Andrew Streitwieser andClayton Heathcock (1981), pages 169-171; and Organic Chemistry, 5^(th)ed., John McMurry (2000), pages 398 and 408; all of which areincorporated herein by reference in their entirety.

Example 45 Structure Activity Relationships

The data set forth in the above-listed Tables illustrate a number ofpreferred embodiments. With regard to Formula II, compounds having ahalogenated substituent at R₁ are preferred and such compounds aregenerally equipotent across the above-described assays. Most preferredare n-halogenated ethyl at R₁.

Also, most preferred are compounds with a hydroxy group at E₅ and theattached carbon is in an S configuration (compounds having thestereochemistry of compound II-18, for example). Oxidation from ahydroxy group to a ketone is less preferred.

In one preferred embodiment, the preferred substituent at R₄ iscyclohexene. In another preferred embodiment, the cyclohexene isoxidized to an epoxide. Less preferred are compounds with hydrogenationof the double bond of the cyclohexene substituent.

Furthermore in some embodiments, preferably, R₃ is methyl, with ethylbeing less preferred.

Example 46 Inhibition of Angiogenesis

Angiogenesis is an important physiological process, without whichembryonic development and wound healing would not occur. However,excessive or inappropriate angiogenesis is associated with a number ofdiseases, conditions, and adverse treatment results. Examples of diseasetypes and conditions associated with excessive angiogenesis includeinflammatory disorders such as immune and non-immune inflammation,rheumatoid arthritis, chronic articular rheumatism and psoriasis;disorders associated with inappropriate or inopportune invasion ofvessels such as diabetic retinopathy, neovascular glaucoma, retinopathyof prematurity, macular degeneration, corneal graft rejection,retrolental fibroplasia, rubeosis, capillary proliferation inatherosclerotic plaques and osteoporosis; and cancer associateddisorders, including for example, solid tumors, tumor metastases, bloodborn tumors such as leukemias, angiofibromas, Kaposi sarcoma, benigntumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas,and pyogenic granulomas, as well as other cancers which requireneovascularization to support tumor growth. Additional examples ofangiogenesis-dependent diseases include, for example, Osler-WebberSyndrome; myocardial angiogenesis; plaque neovascularization;telangiectasia; hemophiliac joints and wound granulation. Furthermore,excessive angiogenesis is also associated with clinical problems as partof biological and mechanical implants (tissue/organ implants, stents,etc.). The instant compositions can be used to inhibit angiogenesis, andthus in the treatment of such conditions. Other diseases in whichangiogenesis plays a role, and to which the instant compounds andcompositions can be used, are known by those of skill in the art.

Particular discussion of angiogenesis in a number of pathophysiologicalconditions such as cancer, rheumatoid arthritis, diabetic retinopathy,age related macular degeneration, endometriosis and obesity is found inFolkman J.(1985) Tumor angiogenesis. Adv Cancer Res. 1985;43:175-203.;Folkman, J. (2001). Angiogenesis-dependent diseases. Semin Oncol, 28,536-42.; Grosios, K., Wood, J., Esser, R., Raychaudhuri, A. & Dawson, J.(2004). Angiogenesis inhibition by the novel VEGF receptor tyrosinekinase inhibitor, PTK787/ZK222584, causes significant anti-arthriticeffects in models of rheumatoid arthritis. Inflamm Res, 53, 133-42;Hull, M. L., Charnock-Jones, D. S., Chan, C. L., Bruner-Tran, K. L.,Osteen, K. G., Tom, B. D., Fan, T. P. & Smith, S. K. (2003).Antiangiogenic agents are effective inhibitors of endometriosis. J ClinEndocrinol Metab, 88, 2889-99; Liu, L. & Meydani, M. (2003).Angiogenesis inhibitors may regulate adiposity. Nutr Rev, 61, 384-7;Mousa, S. A. & Mousa, A. S. (2004). Angiogenesis inhibitors: current &future directions. Curr Pharm Des, 10, 1-9. Each of the above-describedreferences is incorporated herein by reference in its entirety.

The compounds disclosed herein inhibit angiogenesis. The other compoundsdisclosed herein are tested in a transwell migration assay and inhibitmigration. The compounds block vascular endothelial growth-factor(VEGF)-induced migration of multiple myeloma cells in a transwellmigration assay.

The compounds disclosed herein show angiogenesis inhibitory activity inany of various other angiogenesis tests and assays, including one ormore of the following.

The compounds disclosed herein show anti-angiogenic activity in variousother in vitro and in vivo assays. Some examples include: in vitroassays for the evaluation of anti-angiogenesis compounds include,1) themodified Boyden chamber assay which assesses the migration ofendothelial cells in response to pro-angiogenic factors (Alessandri G,Raju K, Gullino P M. (1983) “Mobilization of capillary endothelium invitro induced by effectors of angiogenesis in vivo” Cancer Res.43(4):1790-7.), 2) differentiation assays such as the Matrigel assay inwhich the attachment, migration and differentiation of endothelial cellsinto tubules is analyzed (Lawley T J, Kubota Y. (1989). Induction ofmorphologic differentiation of endothelial cells in culture. J InvestDermatol.August;93(2 Suppl):59S-61S) and 3) organ culture assays inwhich the outgrowth of endothelial (and other cells) is monitored(Nicosia R F, Ottinetti A.(1990). Growth of microvessels in serum-freematrix culture of rat aorta. A quantitative assay of angiogenesis invitro. Lab Invest. July;63(1):115-22.). Some in vivo assays for theevaluation of angiogenesis inhibitors are 1) sponge implantation assays,during which sponges containing cells and/or angiogenic factors and thetest substance are implanted subcutaneously in animals for study of invivo angiogenesis (Plunkett M L, Hailey J A. (1990). An in vivoquantitative angiogenesis model using tumor cells entrapped in alginate.Lab Invest. April 1990;62(4):510-7), 2) the chick chorioallantoicmembrane assay in which test compounds are inserted through a window,cut in the eggshell. The lack of a mature, immune system in the 7-8 dayold chick embryo allows for the study of tumor-induced angiogenesis(Folkman J.(1985) Tumor angiogenesis. Adv Cancer Res. 1985;43:175-203.)and 3) various tumor models in which specific histological analyses canbe used to examine the effect on blood vessels, such as vascular density(CD31/CD34 staining), blood flow and concomitant tumornecrosis/apoptosis (TUNEL staining). In vitro assay examples includeendothelial cell tests (HUVEC (human umbilical vein endothelial cell),aortic, capillary); endothelial cell proliferation assays; endothelialcell DNA synthesis assays; endothelial cell outgrowth assays (Aorticring); endothelial cell migration assays (mentioned above; chemokinesis(colloidal gold), chemotaxis (Boyden chamber)); endothelial cell tubeformation assays; endothelial apoptosis assays; endothelial cellviability assays (trypan blue); angiogenesis factor-transfectedendothelial cell lines; and magnetized microbeads on endothelial cells.Each of the references in this paragraph is incorporated herein byreference in its entirety.

Examples of in vivo assays include transparent chamber tests (e.g.,rabbit ear, hamster cheek, cranial window, and dorsal skin); matriximplants (e.g., subcutaneous injection using sodium alginate,subcutaneous disc (polyvinyl foam implant), rat dorsal air sac, spongeimplant); cornea micropocket assays, for example in rabbits and otherrodents; anterior eye/iris chamber implant assays, mice and knock-outassays; ameroid constriction (heart) in pig and dog; rabbit hindlimbischemia tests; vascularization into tissue (intradermal inoculation,peritoneal cavity/omentum with implant; tumor implants, for example inrabbits, mice or rats.

Also, ex vivo assays are performed using the disclosed compounds.Examples include CAM (chick chorioallantoic membrane assay) and verticalCAM with polymer gel. Immunoassays such as serum assays, urine assayscerebrospinal fluid assays and tissue immunohistochemical assays.

Some of the above assays are described in the following papers, each ofwhich is incorporated herein by reference in its entirety. Grant et al.,In Vitro Cell Dev. Biol. 27A:327-336 (1991); Min et al., Cancer Res.56:2428-2433 (1996); Schnaper et al., J. Cell. Physiol. 165:107-118(1995); Schnaper et al., J. Cell. Physiol. 165:107-118 (1995); Oikawa etal., Cancer Lett. 59:57-66 (1991).

Embodiments relate to methods of using the compounds and compositionsdescribed herein, alone or in combination with other agents, to inhibitangiogenesis and to treat or alleviate diseases and conditionsassociated with excessive or inappropriate angiogenesis. Preferably, theinhibition occurs in connection with vascularization in connection witha disease associated with angiogenesis, such as cancer or any of theother diseases described above, and those that are known by those ofskill in the art. The compounds and compositions can be delivered in anappropriate inhibitory amount. Inhibitory amount is intended to mean anamount of a compound or composition required to effect a decrease in theextent, amount or rate of neovascularization when administered to atissue, animal or individual. The dosage of compound or compositionrequired to be therapeutically effective will depend, for example, onthe angiogenesis-dependent disease to be treated, the route and form ofadministration, the potency and big-active half-life of the moleculebeing administered, the weight and condition of the tissue, animal orindividual, and previous or concurrent therapies. The appropriate amountapplication of the method can be determined by those skilled in the art,using the guidance provided herein. For example, the amount can beextrapolated from in vitro or in vivo angiogenesis assays describedabove. One skilled in the art will recognize that the condition of thepatient needs to be monitored throughout the course of therapy and thatthe amount of the composition administered can be adjusted accordingly.

The present compounds and compositions can be and are used as well inconjunction with other angiogenesis inhibitors. Angiogenic inhibitorsare known in the art and can be prepared by known methods. For example,angiogenic inhibitors include integrin inhibitory compounds such as.alpha-V-beta-3 (αVβ3) integrin inhibitory antibodies, cell adhesionproteins or functional fragments thereof which contain a cell adhesionbinding sequence. Additional angiogenic inhibitors include, for example,angiostatin, functional fragments of angiostatin, endostatin, fibroblastgrowth factor (FGF) inhibitors, FGF receptor inhibitors, VEGFinhibitors, VEGF receptor inhibitors, vascular permeability factor (VPF)inhibitors, VPF receptor inhibitors, thrombospondin, platelet factor 4,interferon-alpha, interferon-gamma, interferon-inducible protein 10,interleukin 12, gro-beta, and the 16 kDa N-terminal fragment ofprolactin, thalidomide, and other mechanisms for the inhibition ofangiogenesis.

Thus, the methods can include the step of administering a compound orcomposition to an animal suffering from a condition associated withexcessive angiogenesis. The methods can further include administeringthe instant compound or composition along with another anti-angiogenesisdrug or along with other therapies for the condition be treated (e.g.,with a chemotherapeutic or immunotherapeutic to treat cancer).

The compounds or compositions can be delivered in any disease and/orpatient appropriate manner. Examples include, intravenous, oral,intramuscular, intraocular, intranasal, intraperatoneal, and the like.

The following references provide additional teaching regarding methodsof using, administering and assaying for angiogenesis inhibition:Angiogenesis Protocols (Methods in Molecular Medicine) by J. CliffordMurray, Humana Press (Mar. 15, 2001) ISBN: 0896036987; TumourAngiogenesis, by R. J. Bicknell, Claire E. Lewis, Napoleone Fe, OxfordUniversity Press (Sep. 1, 1997) ISBN: 0198549377; and Angiogenesis inHealth and Disease: Basic Mechanisms and Clinical Applications, by GaborM. Rubanyi, Marcel Dekker (Nov. 1, 1999) ISBN: 0824781023. Each book isincorporated herein by reference in its entirety. In particular theprotocols and methods are incorporated herein.

Example 47 Formulation to be Administered Orally or the Like

A mixture obtained by thoroughly blending 1 g of a compound obtained andpurified by the method of the embodiment, 98 g of lactose and 1 g ofhydroxypropyl cellulose is formed into granules by any conventionalmethod. The granules are thoroughly dried and sifted to obtain a granulepreparation suitable for packaging in bottles or by heat sealing. Theresultant granule preparations are orally administered at betweenapproximately 100 ml/day to approximately 1000 ml/day, depending on thesymptoms, as deemed appropriate by those of ordinary skill in the art oftreating cancerous tumors in humans.

The examples described above are set forth solely to assist in theunderstanding of the embodiments. Thus, those skilled in the art willappreciate that the methods may provide derivatives of compounds.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand procedures described herein are presently representative ofpreferred embodiments and are exemplary and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the embodiments disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions indicates the exclusion of equivalents of the features shownand described or portions thereof It is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed can beresorted to by those skilled in the art, and that such modifications andvariations are considered to be falling within the scope of theembodiments of the invention.

1. A compound having the structure of Formula I, and pharmaceuticallyacceptable salts and pro-drug esters thereof:

wherein the dashed line indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ is separately selected fromthe group consisting of a hydrogen, a mono-substituted orpoly-substituted saturated C₁-C₂₄ alkyl, mono-substituted,poly-substituted or unsubstituted variants of the following residues:C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy,cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, aminocarbonyl, aminocarbonyloxy, nitro,azido, phenyl, cycloalkylacyl, arylthio, oxysulfonyl, carboxy, cyano,thio, sulfoxide, sulfone, sulfonate ester, thiocyano, boronic acid,boronic ester, and halogenated alkyl including polyhalogenated alkyl,wherein the mono-substituted or poly-substituted saturated C₁-C₂₄ alkylin R₁ is substituted with one or more substituents selected from thegroup consisting of alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano,halogen, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; wherein thehalogenated alkyl in R₁ is a C₁₋₂₄-alkyl poly-halogenated with fluorine,bromine, chlorine or iodine; or the halogenated alkyl is methyl orC₃₋₂₄-alkyl mono-or poly-halogenated with fluorine, bromine, or iodine;where n is equal to 1 or 2, and if n is equal to 2, then R₁ can be thesame or different, and at least one R₁ is not hydrogen; wherein R₂ isselected from the group consisting of hydrogen, a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy,cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl,arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate ester, thiocyano, boronic acid, boronic ester, andhalogenated alkyl including polyhalogenated alkyl; wherein R₃ isselected from the group consisting of a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy,nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio,oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate esters,thiocyano, boronic acid, boronic ester, and halogenated alkyl includingpolyhalogenated alkyl; wherein each of E₁ and E₃ is a substituted orunsubstituted heteroatom selected from the group consisting of O and S,E₂ is a substituted or unsubstituted N, and E₄ is a substituted orunsubstituted heteroatom selected from the group consisting of O, S, andN; and with the proviso that Formula I is not Compound II-16, whereinCompound II-16 has the structure:


2. A compound having the structure of Formula II, and pharmaceuticallyacceptable salts and pro-drug esters thereof:

wherein the dashed lines indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ is separately selected fromthe group consisting of a hydrogen, a mono-substituted orpoly-substituted saturated C₁-C₂₄ alkyl, mono-substituted,poly-substituted or unsubstituted variants of the following residues:C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy,cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, aminocarbonyl, aminocarbonyloxy, nitro,azido, phenyl, cycloalkylacyl, arylthio, oxysulfonyl, carboxy, cyano,thio, sulfoxide, sulfone, sulfonate ester, thiocyano, boronic acid,boronic ester, and halogenated alkyl including polyhalogenated alkyl,wherein the mono-substituted or poly-substituted saturated C₁-C₂₄ alkylin R₁ is substituted with one or more substituents selected from thegroup consisting of alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano,halogen, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; wherein thehalogenated alkyl in R₁ is a C₁₋₂₄-alkyl poly-halogenated with fluorine,bromine, chlorine or iodine; or the halogenated alkyl is methyl orC₃₋₂₄-alkyl mono-or poly-halogenated with fluorine, bromine, or iodine;where n is equal to 1 or 2, and if n is equal to 2, then R₁ can be thesame or different, and at least one R₁ is not hydrogen; wherein R₃ isselected from the group consisting of a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy,nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio,oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate ester,thiocyano, boronic acid, boronic ester, and halogenated alkyl includingpolyhalogenated alkyl; wherein R₄ is separately selected from the groupconsisting of a hydrogen, a halogen, mono-substituted, poly-substitutedor unsubstituted variants of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio,oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate ester,thiocyano, boronic acid, boronic ester, and halogenated alkyl includingpolyhalogenated alkyl, and m is equal to 1 or 2, and if m is equal to 2,then R₄ can be the same or different; wherein each of E₁ and E₃ is asubstituted or unsubstituted heteroatom selected from the groupconsisting of O and S, E₂ is a substituted or unsubstituted N, and eachE₄ and E₅ are a substituted or unsubstituted heteroatom selected fromthe group consisting of O, S, and N; and with the proviso that FormulaII is not Compound II-16, wherein Compound II-16 has the structure:


3. The compound of claim 2, wherein E₅ is selected from the groupconsisting of OH, O, S, N, NH, NH₂, NOH, NHOH, OR₁₀, SR₁₁, NR₁₂, andNHOR₁₃, wherein R₁₀, R₁₁, R₁₂, and R₁₃ each is separately selected fromthe group consisting of hydrogen, and a substituted or unsubstitutedalkyl, acyl, aryl, and heteroaryl.
 4. The compound of claim 2, whereinE₅ is OH.
 5. The compound of claim 2, wherein each of E₁, E₃ and E₄ is Oand E₂ is NH.
 6. The compound of claim 2, wherein R₃ is methyl.
 7. Thecompound of claim 2, wherein R₄ is an unsubstituted or substitutedcycloalkyl.
 8. The compound of claim 2, wherein at least one R₄ is anunsubstituted or substituted cycloalkyl and E₅ is an oxygen.
 9. Thecompound of claim 2, wherein at least one R₄ is a di-substitutedcyclohexane.
 10. The compound of claim 2, wherein at least one R₄ is asubstituted or an unsubstituted branched C₄-C₂₄ alkyl.
 11. The compoundof claim 2, wherein: at least one R₄ is

wherein R₅ is separately selected from the group consisting of ahydrogen, a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, oxy, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate ester, thiocyano, boronic acid, boronic ester, andhalogenated alkyl including polyhalogenated alkyl, and m′ is 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or 11 and if m′ is more than 1, then R₅ can be thesame or different; and where the substituents R₅ can form a ring. 12.The compound of claim 11, wherein R₃ is methyl.
 13. A compound havingthe structure of Formula I, and pharmaceutically acceptable salts andpro-drug esters thereof:

wherein the dashed line indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ is separately selected fromthe group consisting of a hydrogen, a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarbonyloxy,nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio,oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate ester,thiocyano, boronic acid, boronic ester, and halogenated alkyl includingpolyhalogenated alkyl, where n is equal to 1 or 2, and if n is equal to2, then R₁ can be the same or different, and at least one R₁ is nothydrogen; wherein R₂ is selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, acyl,acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarbonyloxy,nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio,oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate ester,thiocyano, boronic acid, boronic ester, and halogenated alkyl includingpolyhalogenated alkyl; wherein R₃ is selected from the group consistingof a halogen, mono-substituted, poly-substituted or unsubstitutedvariants of the following residues: saturated C₁-C₂₄ alkyl, unsaturatedC₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio,sulfoxide, sulfone, sulfonate ester, thiocyano, boronic acid, boronicester, and halogenated alkyl including polyhalogenated alkyl; whereineach of E₁, and E₃ is a substituted or unsubstituted heteroatom selectedfrom the group consisting of O and S, E₂ is a substituted orunsubstituted N, and E₄ is a substituted or unsubstituted heteroatomselected from the group consisting of O, S, and N; and with the provisothat Formula II is not Compound II-16, wherein Compound II-16 has thestructure:


14. A compound having the structure of Formula II, and pharmaceuticallyacceptable salts and pro-drug esters thereof:

wherein the dashed lines indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ is separately selected fromthe group consisting of a hydrogen, a halogen, mono-substituted,poly-substituted or unsubstituted variants of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarbonyloxy,nitro, azido, phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio,oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone, sulfonate ester,thiocyano, boronic acid, boronic ester, and halogenated alkyl includingpolyhalogenated alkyl, where n is equal to 1 or 2, and if n is equal to2, then R₁ can be the same or different, and at least one R₁ is nothydrogen; wherein R₃ is selected from the group consisting of a halogen,mono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy,cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl,arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,aminocarbonyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate ester, thiocyano, boronic acid, boronic ester, andhalogenated alkyl including polyhalogenated alkyl; wherein R₄ isseparately selected from the group consisting of a hydrogen, a halogen,a mono-substituted or poly-substituted variant of the followingresidues: C₁-C₂₄ saturated alkyl, cycloalkyl, cycloalkenyl and aryl, amono-substituted, poly-substituted or unsubstituted variants of thefollowing residues: unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, acyl,acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, alkoxy, cycloalkoxy,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarbonyloxy, nitro, azido, hydroxy, alkylthio,arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide, sulfone,sulfonate ester, thiocyano, boronic acid, boronic ester, and halogenatedalkyl including polyhalogenated alkyl, and m is equal to 1 or 2, and ifm is equal to 2, then R₄ can be the same or different; wherein themono-substituted or poly-substituted saturated C₁-C₂₄ alkyl, cycloalkyl,cycloalkenyl or aryl in R₄ is substituted with one or more substituentsselected from the group consisting of alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, carboxyl,carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; wherein each ofE₁ and E₃ is a substituted or unsubstituted heteroatom selected from thegroup consisting of O and S, E₂ is a substituted or unsubstituted N, andeach E₄ and E₅ are a substituted or unsubstituted heteroatom selectedfrom the group consisting of O, S, and N; and with the proviso thatFormula II is not Compound II-16, wherein Compound II-16 has thestructure:


15. The compound of claim 14, wherein each of E₁, E₃ and E₄ is O and E₂is NH.
 16. The compound of claim 14, wherein R₃ is methyl.
 17. Thecompound of claim 14, wherein n is equal to 1 or 2, and where n is equalto 2, at least one R₁ is CH₂CH₂X, wherein X is selected from the groupconsisting of H, F, Cl, Br, and I.
 18. The compound of claim 14,wherein: n is equal to 2; at least one of the R₁ substituents ishydrogen and the other R₁ substituent is CH₂CH₂X, wherein X is selectedfrom the group consisting of H, F, Cl Br, and I; R₃ is methyl; at leastone R₄ is a substituted cyclohexane; each of E₁, E₃ and E₄ is O and E₂is NH; and E₅ is OH.
 19. The compound of claim 14, wherein R₁ is asubstituted C₁ to C₅ alkyl.
 20. The compound of claim 14, wherein E₅ isOH.
 21. A pharmaceutical composition comprising at least one compound ofclaim
 14. 22. A compound having the structure of Formula II, andpharmaceutically acceptable salts and pro-drug esters thereof:

wherein the dashed lines indicates that the designated bond is either asingle bond or a double bond, and wherein R₁ is separately selected fromthe group consisting of a hydrogen; a halogen; azidoethyl;thiocyanatoethyl; boronic acid(C₀₋₆ alkyl); boronic ester(C₀₋₆ alkyl);hydroxyethyl; an unsubstituted saturated C₂₋₃ alkyl; a mono-substitutedor poly-substituted C₁₋₂₄ alkyl substituted with one or moresubstituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyloxy, oxyacylamino, cyano, carboxyl, carboxylalkyl, keto,thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,alkoxyamino, nitro, sulfonate ester, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl; and mono-substituted, poly-substituted orunsubstituted variants of the following residues: unsaturated C₂-C₂₄alkenyl, C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, thio, sulfoxide,sulfone, sulfonate ester, thiocyano, boronic acid, boronic ester, andhalogenated alkyl including polyhalogenated alkyl; where n is equal to 1or 2, and if n is equal to 2, then R₁ can be the same or different, andat least one R₁ is not hydrogen; wherein R₃ is selected from the groupconsisting of a halogen, mono-substituted, poly-substituted orunsubstituted variants of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido,phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, thio, sulfoxide, sulfone, sulfonate ester, thiocyano,boronic acid, boronic ester, and halogenated alkyl includingpolyhalogenated alkyl; wherein R₄ is separately selected from the groupconsisting of a hydrogen, and an unsubstituted, a mono-substituted orpoly-substituted cycloalkyl, cycloalkenyl, and aryl, and m is equal to 1or 2, and if m is equal to 2, then R₄ can be the same or different;wherein each of E₁ and E₃ is a substituted or unsubstituted heteroatomselected from the group consisting of O and S, E₂ is a substituted orunsubstituted N, and each E₄ and E₅ are a substituted or unsubstitutedheteroatom selected from the group consisting of O, S, and N; and withthe proviso that Formula II is not Compound II-16, wherein CompoundII-16 has the structure:


23. The compound of claim 22, wherein the compound of Formula II isselected from the group consisting of:

and, wherein R₈ is selected from the group consisting of H, F, Cl, Brand I.
 24. The compound of claim 22, wherein the compound of Formula IIis selected from the group consisting of:

wherein when the compound of Formula II is the compound II-3 9 or thecompound II-40, then R is selected from the group consisting of H,unsubstituted alkyl, substituted alkyl, unsubstituted aryl andsubstituted aryl;

wherein: when the compound of Formula II is the compound II-42, then nis 0, 1, 2, 3, 4, 5 or 6, and each R is independently selected from thegroup consisting of H and alkyl.
 25. A compound having the followingstructure, and pharmaceutically acceptable salts and pro-drug estersthereof:

wherein R₈ is selected from the group consisting of fluorine, chlorine,bromine and iodine.
 26. The compound of claim 25, where R₈ is chlorine.